Replication-competent controlled alpha-herpesvirus vectors and uses therefore

ABSTRACT

The present disclosure relates to replication-competent controlled herpesviruses whose transient replication in a desired inoculation site region of a subject can be activated by the delivery of an appropriate heat dose to the inoculation site region. In related recombinant viruses, activation requires delivery of a heat dose in the presence in the inoculation site region of an effective concentration of a small-molecule regulator. The viruses are further engineered to be capable of replicating efficiently in the desired inoculation site region but essentially not in nerve ganglia and other nerve cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of European Patent Application No.18207121.7, filed 19 Nov. 2018, which application is incorporated hereinby reference in its entirety.

REFERENCE TO THE SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled“VIR4PCT-ST25-replace.txt”, created Feb. 7, 2023, which is 12,007 bytesin size. The information in the electronic format of the SequenceListing is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present invention relates to certain replication-competentcontrolled alpha-herpesviruses and their utilization, including forimmunization and cancer therapy.

BACKGROUND OF THE INVENTION

Replication-competent controlled viruses are viruses whose replicationis under deliberate control. They are capable of transiently replicatingupon activation but are essentially nonreplicating in the absence ofactivation. The present disclosure is concerned with certainreplication-competent controlled viruses (abbreviated herein as RCCHVsfor replication-competent controlled herpesviruses) having particularbeneficial properties. These RCCHVs are derived from virulent viruses ofthe alpha-herpesvirus subfamily, in particular mammalian HSV-1, HSV-2and vaccinia viruses. While the primary targets of the latter virusesare mucoepithelial cells, their tropism is much broader. The viruseshave a lytic phase during symptomatic infection as well as a latentphase where they lie dormant in sensory and cranial nerve ganglia butremain susceptible to reactivation.

Replication-competent controlled viruses were described previously. Forexample, Chong et al. (2002) described a complementing pair ofadenoviruses, of which one virus expressed a gene for arapamycin-controlled heterologous transactivator and the other containedan E1A gene that had been brought under the control of atransactivator-responsive promoter (Mol Ther (2002) 5: 195-203). Otherreplication-competent controlled viruses and virus pairs were disclosedIn U.S. Pat. Nos. 7,906,312 and 8,137,947, respectively. In the RCCHVsdescribed by Bloom et al. (2015) (J Virol (2015) 89: 10668-10679), asmall-molecule regulator-controlled heterologous transactivator wasexpressed under the control of a promoter cassette comprising a heatshock promoter and a transactivator-responsive promoter, and theresident promoters of one or more replication-essential genes werereplaced by transactivator-responsive promoters.

RCCHVs may be employed as vaccines or vaccine vectors. As demonstratedin Bloom et al. (2018), the efficient replication of activated RCCHVssubstantially enhanced immune responses to the vectors or toheterologous antigens expressed by the vectors when compared tounactivated vectors or control viruses, respectively ((J Virol (2018)92: e00616-18); see also International Patent Application PublicationWO2016/030392). RCCHVs may also be used in oncolytic or other therapies.

SUMMARY OF THE INVENTION

The present disclosure relates to replication-competent controlledalpha-herpesvirus-derived viruses (RCCHVs) whose replication can betransiently activated in certain infected nonneural cells but thatcannot be so activated in infected sensory or other neural cells.

Subsequent to administration to a tissue region of the body of amammalian subject (also referred to as the inoculation site region), aheat-controlled RCCHV of this disclosure is induced to replicate by alocalized administration of an appropriate heat dose to the inoculationsite region. Heat can be focused. Hence, the virus can be activated toreplicate only in the inoculation site region, minimizing anypathological effects that are known to be associated with disseminatedreplication of the wildtype virus. Subsequent to its heat activation,the virus will replicate with a high efficiency in the heated region.This replication is transient and ends with the lysis of the infectedcells in the heated region. Hence, virus replication is also limited intime/extent, further decreasing the possibility of untoward pathologicaleffects. Over the ensuing weeks, the virus is cleared from the body ofthe mammalian subject, except from sensory and, depending on the virusadministration site, cranial nerve ganglia where it survives in a latentform. Virus reactivation which may otherwise occur if the mammaliansubject is exposed to a severe physiological stress (high fever) isprevented because a replication-essential gene of the RCCHV has beenplaced under the control of a tissue-selective promoter that isessentially inactive in nerve ganglia of the mammalian subject. In thecase of a heat- and small-molecule regulator (SMR)-controlled RCCHV,replication in the inoculation site region of a mammalian subject istriggered by a localized heat treatment in the presence of an effectiveconcentration of the appropriate SMR. The efficient but transientreplication ends when the concentration of the SMR has fallen to anineffective level in the inoculation site region (as a result ofelimination by normal physiological processes) or when all infected (andheat-exposed) cells have been lysed (whichever occurs first).Thereafter, in the absence of a re-administration of a dose of the SMRthat suffices to co-activate the virus, the subject is expected to beprotected from reactivation. However, in the absence of theabove-described protective mechanism, there is no reason for believingthat reactivation could not occur upon re-administration of an effectiveconcentration of the SMR at a time at which the mammalian subject isalso experiencing a stress. An SMR will be susceptible to at leastsporadic re-administration to certain subjects, if it has an approvedpharmacological activity. This is more than a theoretical possibility asSMRs of choice preferably are well known molecules that are readilyavailable, have been tested in human toxicological studies and, ideally,are approved for human use.

An RCCHV of the present disclosure is a recombinant herpesvirus thatcomprises a first heterologous promoter that is a heat shock promoter ornucleic acid sequence that functions as a heat shock promoter. The term“heterologous” is meant to indicate that the genetic element it relatesto is not identical with any genetic element of the herpesvirus thatserves as the backbone for the construction of the RCCHV. The firstheterologous promoter is employed to control, directly or indirectly asis discussed below, the expression of a first replication-essentialgene.

In a first embodiment, the first heterologous promoter is inserted inthe genome of the backbone herpesvirus so as to replace the resident(viral) promoter of the first replication-essential viral gene. Hence,the first replication-essential gene is placed under the control of thefirst heterologous promoter.

In a second embodiment, the first heterologous promoter controls theexpression of a gene for a heterologous transactivator, whereby acassette comprising the first heterologous promoter and the functionallylinked transactivator gene is inserted in a suitable intergenic regionin the genome of the backbone herpesvirus. A transactivator-responsivepromoter is inserted in the genome of the virus so as to replace theresident promoter of the first replication-essential viral gene. Hence,the first replication-essential gene is placed under the control of thetransactivator. In a variation of the second embodiment, the firstheterologous promoter is a nucleic acid sequence to functions as a heatshock promoter as well as a transactivator-responsive promoter. Thetransactivator can be any unregulated transactivator that beginsmediating transcription from a transactivator-responsive promoter in amammalian cell as soon it (the transactivator) is synthesized in thecell. Preferred is a transactivator that does not affect or onlyminimally affects host gene expression in a mammalian host. Thetransactivator can also be a regulated transactivator that is incapableof mediating transcription from a transactivator-responsive promoter ina mammalian cell prior to an activating interaction with an appropriateSMR. An RCCHV of the present disclosure that carries a gene for anSMR-activated transactivator is referred to as a heat- andSMR-controlled RCCHV, and an RCCHV that does not express a regulatedtransactivator is referred to as a heat-controlled RCCHV. In principle,the regulated transactivator can be any transactivator that can beactivated by an SMR. Preferred is a transactivator that does not affector only minimally affects host gene expression in a mammalian host andfor which an SMR is available that has no undue toxicity in themammalian subject to which RCCHVs are to be administered. As discussedfurther below, there are several transactivator/SMR combinations thatmay satisfy the latter requirements. Preferred is a regulatedtransactivator that contains a truncated ligand-binding domain from aprogesterone receptor and is activated by a progesterone receptorantagonist (antiprogestin) or other molecule capable of interacting withthe ligand-binding domain and of activating the transactivator. Typicalfor the class of antiprogestins that activate the latter transactivatorare mifepristone and ulipristal. The most preferred transactivator isGLP65. Burcin et al. (1999) (Proc Natl Acad Sci USA (1999) 96: 355-60);Ye et al. (2002) (Meth Enzymol (2002) 346: 551-61).

An RCCHV of the present disclosure further comprises a secondheterologous promoter that replaces the resident promoter of a secondreplication-essential viral gene. Hence, the secondreplication-essential viral gene has been placed under the control ofthe second heterologous promoter. The second heterologous promoter is apromoter that is known to be active in cells in the intended inoculationsite region of a mammalian subject (i.e., the region to which the RCCHVis to be administered) but is known to be essentially inactive in thecells of the nerve ganglia of the mammalian subject. The terms “known tobe active” and “known to be essentially inactive” refer to transcriptlevels of the gene that is naturally controlled by the chosen secondheterologous promoter as published in professional databases, of whichBioGPS is a preferred database (biogps.org). A suitable secondheterologous promoter is a promoter of a mammalian gene whose transcriptlevel in tissue regions that are intended for administration of an RCCHV(inoculation site regions) is at least about 50 times, more preferablyat least about 100 times and most preferably at least about 500 timeshigher than in (cells of) nerve ganglia or other nerve tissue. Inaddition, the transcript level of the latter mammalian gene in nerveganglia or other nerve tissue is less than about three times the mediantranscript level of all tissues examined. Preferably, it is close to orless than the median transcript level of all tissues examined. It isnoted that a transcript level corresponding to the median transcriptlevel of a gene that is expressed in a highly tissue-specific fashioncorresponds to a level that is close to or at the limit of detection,and a transcript level of three times the median transcript level isstill extremely low. Hence, the promoter of such a gene is “essentiallyinactive”.

The primary targets of the alpha-herpesvirues that can serve asbackbones for the construction of RCCHVs of the present disclosure aremucoepithelial cells. Hence, a preferred inoculation site region of anRCCHV of the present disclosure can be a cutaneous or subcutaneousregion, or a mucosal membrane of a mammalian subject. A particularlypreferred inoculation site region may be an epidermal region, preferablyan epidermal region on an extremity of a mammalian subject. With thesepreferences, the databases may be mined for genes that are active in theepidermis (or the skin) but are essentially inactive in nerve ganglia,e.g., the dorsal root ganglia. Such a search will uncover, e.g., thekeratin-1 gene as is discussed further below. The promoter of this genemay serve as a second heterologous promoter for an RCCHV that isintended for administration to a skin region of a mammalian subject.

An RCCHV of the present disclosure can be derived from a (wildtype)herpes simplex virus 1 (HSV-1), a herpes simplex virus 2 (HSV-2), or avaricella zoster virus (VZV).

In an RCCHV of the present disclosure that has been derived from anHSV-1 or HSV-2, the viral gene ICP47 may be deleted or renderednonfunctional. The product of this gene binds to transporters associatedwith antigen processing (TAP), interfering with the presentation ofantigens to MHC class I molecules and, consequently, with immunerecognition by cytotoxic T-lymphocytes.

An RCCHV of the present disclosure can be engineered to carry in itsgenome an expressible gene (or parts thereof) from another pathogen, anexpressible heterologous gene encoding an immune-modulatory polypeptideor an expressed heterologous gene encoding another polypeptide or anycombination of one or more of such genes (or gene portions). The latterheterologous genes (i.e., genes not originally present in the wildtypevirus from which the RCCHV was derived) can be placed under the controlof any suitable promoter, including a constitutively active viral ornon-viral promoter, the first heterologous promoter (directly orindirectly as discussed above) or the second heterologous promoter. Thegene for another pathogen can be a gene encoding an influenza virussurface antigen or internal protein or parts thereof (representingselected polypeptide regions). It can also be a gene encoding a humanimmunodeficiency virus surface antigen or internal protein or partsthereof (representing selected polypeptide regions).

Also encompassed by the present disclosure are vaccine compositions orcompositions for cancer therapy or genetic therapy which compositionscomprise an effective amount of an RCCHV of the present disclosure and apharmaceutically acceptable carrier or excipient. In the case of a heat-and SMR-controlled RCCHV, a composition comprising an effective amountof an SMR that is capable of activating the heterologous transactivatorcan be co-administered with any one of the latter compositions or can beadministered separately, including by a different route. For example, avaccine composition can be administered topically to a region of theskin of a mammalian subject and an SMR-comprising composition may beadministered systemically (e.g., per os). Alternatively, a vaccinecomposition or a composition for cancer therapy or for genetic therapycomprising an effective amount of an RCCHV and a pharmaceuticallyacceptable carrier or excipient may further comprise an effective amountof an SMR that is capable of activating the transactivator comprised inthe RCCHV.

Also encompassed by the present disclosure are any uses of RCCHVs of thepresent disclosure (or of compositions comprising an effective amount ofan RCCHV of the present disclosure and a pharmaceutically acceptablecarrier or excipient), including for therapeutic or prophylacticvaccination of mammalian subjects. Vaccine uses can relate tovaccination against the virus from which the RCCHV was derived or, if aheterologous antigen of another pathogen is expressed from the RCCHV,against that other pathogen. Alternatively, uses may concern treatment,including oncolytic treatment, of a cancer (e.g., a melanoma) in amammalian subject. Furthermore, they may relate to a genetic treatmentof a condition or disease that responds to a protein expressed from aheterologous gene carried by the RCCHV.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 presents a nucleotide sequence for the promoter and RNA leaderregion of the mouse keratin 1 (KRT1) gene (retrieved from the EukaryoticPromoter Database EPD (epd.epfl.ch)). The sequence includes 1200nucleotide of 5′ untranscribed sequence and the first 200 nucleotides ofthe transcript. The start of transcription site is bolded.

FIG. 2 presents a nucleotide sequence for the promoter and RNA leaderregion of the mouse keratin 77 (KRT77) gene (retrieved from theEukaryotic Promoter Database EPD (epd.epfl.ch)). The sequence includes1200 nucleotide of 5′ untranscribed sequence and the first 200nucleotides of the transcript. The start of transcription site isbolded.

FIG. 3 shows the nucleotide sequence of human HSF1 present in plasmidCMV-hHSF1. The protein-coding sequence is underlined.

DETAILED DESCRIPTION

Unless otherwise defined below or elsewhere in the presentspecification, all terms shall have their ordinary meaning in therelevant art.

“Replication of virus” or “virus/viral replication” are understood tomean multiplication of viral particles. Replication is often measured bydetermination of numbers of infectious virus, e.g., plaque-forming unitsof virus (pfu). However, replication can also be assessed by biochemicalmethods such as methods that determine amounts of viral DNA, e.g., by areal-time PCR procedure, levels of viral gene expression, e.g., byRT-PCR of gene transcripts, etc. However, it is understood that marginalincreases in levels of viral DNA or viral gene transcripts or proteinproducts may not translate in corresponding marginal increases in virusreplication due to threshold effects.

A “small-molecule regulator” or “SMR” is understood to be a lowmolecular weight ligand of a transactivator used in connection with thisinvention. The SMR is capable of activating the transactivator. The SMRis typically, but not necessarily, smaller than about 1000 Dalton (1kDa).

The term “transactivator” or “heterologous transactivator” is usedherein to refer to a non-viral and, typically, engineered transcriptionfactor that can positively affect transcription of a gene controlled bya transactivator-responsive promoter.

A “small-molecule regulator (SMR)-activated transactivator” or“heterologous small-molecule regulator (SMR)-activated transactivator”is a non-viral and, typically, engineered transcription factor that whenactivated by the appropriate small-molecule regulator (SMR) positivelyaffects transcription of a gene controlled by atransactivator-responsive promoter.

A “transactivator-responsive promoter” is a promoter that contains oneor more sequence elements that can be bound by a transactivator and thatis essentially inactive prior to being bound by an active transcriptionfactor.

“Activated” when used in connection with a transactivated gene meansthat the rate of expression of the gene is measurably greater afteractivation than before activation or, when used in connection with acontrollable heterologous promoter means that thetranscription-enhancing activity of the promoter is measurably greaterafter activation than before activation. When used in connection with aSMR-activated transactivator, “active” or “activated” refers to atransactivation-competent form of the transactivator. The transactivatoris rendered transactivation-competent by the binding of the appropriateSMR.

Herein, a virus, whose genome includes a foreign (heterologous)non-viral or viral gene, is either referred to as a “virus” or a “viralvector”.

A “replication-competent controlled virus” is a recombinant virus whosereplicative ability is under deliberate control (i.e., whose replicationcan be triggered by a deliberate intervention). In such a virus,replication of at least one replication-essential viral gene is underthe control of a gene switch that can be deliberately activated. A“replication-competent controlled herpesvirus”, abbreviated as “RCCHV”,is a replication-competent controlled virus that has been derived from awildtype herpesvirus.

A “recombinant virus” or “recombinant” refers to a virus that has beenaltered by an experimenter.

A “heterologous promoter” is a promoter that, as a whole, does notnaturally occur in the wildtype virus that serves as the backbone forthe construction of a replication-competent controlled virus thatcontains the heterologous promoter. The possibility that such aheterologous promoter also contains elements of a viral promoter isencompassed by the term.

A “replication-essential gene” or a “gene required for efficientreplication” is arbitrarily defined herein as a viral gene whose loss offunction diminishes replication efficiency by a factor of ten orgreater. Replication efficiency can be estimated, e.g., in a(single-step) growth experiment. For many viruses it is well known whichgenes are replication-essential genes. For herpesviruses see, e.g.,Nishigawa (1996) (Nagoya L Med Sci (1996) 59: 107-19).

An “effective amount of a replication-competent controlled herpesvirus(RCCHV)” is an amount of virus that upon single or repeatedadministration to a subject followed by activation confers a therapeuticeffect on the treated subject, at a reasonable benefit/risk ratioapplicable to any medical treatment. When used in the context ofprophylaxis or prevention, an “effective amount” of an RCCHV of thedisclosure is meant to be an amount which, when administered (andthereafter activated) once or multiple times over the course of aprophylactic or preventative (e.g., vaccination) regime, confers adesired prophylactic effect on the treated subject. In general, what isan “effective amount” will vary depending on the route of administrationas well as the possibility of co-usage with other agents. It will beunderstood, however, that the total or fractional dosage of compositionsof the present disclosure comprising an RCCHV will be decided by theattending physician within the scope of sound medical judgment. Thespecific effective dose level for any particular subject may be adjustedbased on a variety of factors including the disorder being treated andthe severity of the disorder; the activity of the RCCHV employed; thespecific composition employed; the age, body weight, general health, sexand diet of the patient; the time of administration, route ofadministration, and rate of elimination of the specific RCCHV employed;the duration of the treatment; drugs used in combination orcontemporaneously with the RCCHV employed; and like factors well knownin the medical arts. The latter factors will be considered in thecontext of therapeutic applications of replication-competent controlledviruses as well as in the context of prophylactic or preventativeapplications.

An “effective amount of a small-molecule regulator (SMR)” is an amountthat when administered to a subject by a desired route is capable ofco-activating a heat- and SMR-controlled RCCHV of the present disclosurewith which the subject concurrently is, has been or will be inoculatedto undergo at least one round of replication in the administration siteregion (also referred to as “inoculation site region”).

A “subject” or a “mammalian subject” is a mammalian animal or a humanperson.

“Promoter of a heat shock gene”, “heat shock gene promoter” and “heatshock promoter” are used synonymously. A “nucleic acid that acts as aheat shock promoter” can be a heat shock promoter or a nucleic acid thatcontains sequence elements of the type present in heat shock promoterswhich elements confer heat activation on a functionally linked gene.

A “heat shock gene” is defined herein as any gene, from any eukaryoticorganism, whose activity is enhanced when the cell containing the geneis exposed to a temperature above its normal growth temperature.Typically, such genes are activated when the temperature to which thecell is normally exposed is raised by 3-10° C. Heat shock genes comprisegenes for the “classical” heat shock proteins, i.e., HSP110, HSP90,HSP70, HSP60, HSP40, and HSP20-30. They also include otherheat-inducible genes such as genes for MDR1, ubiquitin, FKBP52, hemeoxidase and other proteins. The promoters of these genes, the “heatshock promoters”, contain characteristic sequence elements referred toas heat shock elements (HSE) that consist of perfect or imperfectsequence modules of the type NGAAN or AGAAN, which modules are arrangedin alternating orientations (Amin et al. (1988) (Mol Cell Biol (1988) 8:3761-3769); Xiao and Lis (1988) (Science (1988) 239: 1139-1142);Fernandes et al. (1994) (Nucleic Acids Res (1994) 22: 167-173). Theseelements are highly conserved in all eukaryotic cells such that, e.g., aheat shock promoter from a fruit fly is functional and heat-regulated ina frog cell (Voellmy and Rungger (1982) (Proc Natl Acad Sci USA (1982)79: 1776-1780). HSE sequences are binding sites for heat shocktranscription factors (HSFs; reviewed in Wu (1995) (Annu Rev Cell DevBiol (1995) 11: 441-469). The factor primarily responsible foractivation of heat shock genes in vertebrate cells exposed to heat or aproteotoxic stress is heat shock transcription factor 1 (abbreviated as“HSF1”) (Baler et al. (1993) (Mol Cell Biol (1993) 13: 2486-2496);McMillan et al. (1998) (J Biol Chem (1998) 273: 7523-7528). Preferredpromoters for use in RCCHVs discussed herein are those of inducibleHSP70 genes. A particularly preferred heat shock promoter is thepromoter of the human HSP70B gene (Voellmy et al. (1985) (Proc Natl AcadSci USA (1985) 82: 4949-4953).

“Vaccine” typically refers to compositions comprising microorganismsthat are killed, replication-defective or otherwise attenuated. Herein,the term is expanded to also include compositions comprising RCCHVs thatcan induce an immune response in the subject to which they areadministered.

Current thought appears to be that in order to be effective or moreeffective, respectfully, improved vaccine candidates for preventing ortreating diseases such as herpes, HIV, tuberculosis or influenza need toelicit a balanced immune response that also includes a powerful effectorT cell response. The present invention relates to RCCHVs that, uponactivation, replicate with efficiencies that approach those of therespective wild type viruses. It is hypothesized that these recombinantviruses and any heterologous protein they express will be potentimmunogens that elicit balanced immune responses.

To obtain an RCCHV of the present disclosure, a wild type HSV-1, HSV-2or varicella zoster virus (VZV) is genetically altered by placing atleast one replication-essential viral gene under the control of a geneswitch that has a broad dynamic range, i.e., that essentially functionsas an on/off switch. The gene switch can be a highly heat-inducible heatshock promoter or a nucleic acid sequence that functions as such a heatshock promoter (the promoter also being referred to herein as “firstheterologous promoter”). Alternatively, it can be a highlyheat-inducible heat shock promoter (or nucleic acid sequence thatfunctions as such a heat shock promoter) that drives the expression of agene for a heterologous transactivator, and the heterologoustransactivator controls the expression of the replication-essential genethat is functionally linked to a transactivator-responsive promoter. Asheat shock promoters can only be transiently activated, it may beadvantageous to include an element that permits autoactivated synthesisof the transactivator. Hence, the transactivator gene may be controlledby a nucleic acid sequence that acts both as a heat shock promoter andas a transactivator-responsive promoter. Such a nucleic acid sequence ispresent in several of the exemplified RCCHVs and is specificallydiscussed in Example 2 that relates to the construction of HSV-GS1/3.See also FIG. 3 in Vilaboa et al. (2005) (Mol Ther (2005) 12: 290-298).

Any transactivator may be employed as long as it is transcriptionallycompetent when synthesized in a mammalian cell (i.e., is constitutivelyactive) and comprises a DNA-binding domain that specifically binds to aDNA sequence element that is present in a transactivator-responsivepromoter. Preferred is a transactivator that only minimally affects theexpression of the resident genes in cells of a subject that are targetedby the RCCHV, i.e., that has no undue toxicity in these cells.Advantageously, the transactivator is an SMR-activated transactivatorthat is active in the presence of the SMR but is essentially inactive inits absence. To construct an RCCHV that relies on a highlyheat-inducible heat shock promoter (or nucleic acid sequence thatfunctions as such a heat shock promoter) as the gene switch, theresident promoter of at least one replication-essential gene in awildtype herpesvirus is replaced with the latter heat shock promoter. Toconstruct an RCCHV in which one or more replication-essential genes arecontrolled by a transactivator that is expressed from a heat shockpromoter-driven gene, a wildtype herpesvirus is modified to contain theexpressible gene for the transactivator in a location in the viralgenome in which an insertion of such a gene does not interfere withvirus function, in particular with replication efficiency. The U L43/44and UL37/38 intergenic regions of HSV-1 are such locations. Thepromoters of the one or more replication-essential genes are replaced bytransactivator-responsive promoters. The resulting recombinant virusesare referred to as heat-controlled or, when co-activated by an SMR, asheat- and SMR-controlled RCCHVs.

Wildtype HSV-1, HSV-2 or VZV are known to preferentially targetmucoepithelial cells and to establish latency in cells of nerve ganglia,e.g., in cells of the dorsal root ganglia. The preferred RCCHVs of thepresent disclosure are engineered to replicate efficiently inmucoepithelial cells (and derived cells), e.g., epidermal cells of theskin, but, to prevent reactivation from latency, to not (or onlyminimally) replicate in cells of nerve ganglia. This is achieved by theuse of an appropriate tissue- or cell type-specific or restricted(heterologous) promoter for controlling the expression of a furtherreplication-essential gene. Hence, the construction of an RCCHV of thepresent disclosure involves replacement in the above-describedheat-controlled or heat- and SM R-controlled RCCHV of the residentpromoter of the further replication-essential gene with such a tissue-or cell type-specific or restricted (heterologous) promoter. It isunderstood that an RCCHV of the present disclosure can also beconstructed by first replacing in a wildtype herpesvirus the promoter ofa replication-essential gene with a tissue- or cell type-specificpromoter and subsequently placing at least one furtherreplication-essential gene under heat or heat and SMR control.

A tissue- or cell type-specific or restricted promoter that isappropriate for the intended use in an RCCHV of the present disclosurecan be identified by mining available databases and other scientificliterature (see, e.g., the BioGPS database or the article of Su et al.(2002) (Proc Natl Acad Sci USA (2002) 99: 4465-4470).

For an RCCHV of the present disclosure that is to be used as a vaccineor a vaccine vector and is to be administered to a region of the skin ofa human subject, a promoter that is highly active in all epidermallayers of the human skin but not (or only minimally) in cells of nerveganglia may be selected for driving the expression of one or morereplication-essential viral genes. A particular promoter that may beemployed is the human keratin 1 (KRT1) promoter. Edqvist et al. (2015)(J Histochem Cytochem (2015) 63: 129-41). Inspection of the BioGPSdatabase (biogps.org) reveals that this promoter is essentially onlyexpressed in skin (and other epithelia as discussed below). Noexpression is evident in nerve ganglia (Table 1). Another usefulpromoter is that of the human KRT10 gene. Transcript levels are farhigher in the skin than in any other tissue/organ. Essentially noexpression occurs in nerve ganglia. For an RCCHV vaccine to beadministered to the skin of a murine subject, a suitable promoter may bethat of the mouse KRT77 gene. The latter gene is highly active in themouse epidermis but not in any other adult tissue. Essentially noexpression occurs in the dorsal root ganglia. Other useful promoters mayby the mouse KRT1 and KRT10 promoters for which essentially no activityhas been demonstrated in nerve ganglia. That they are also active in thestomach may not significantly detract from their usefulness in vaccineapplications. The nucleotide sequences of the latter promoters can befound in the Eukaryotic Promoter Database (epd.epfl.ch) and elsewhere.

For an RCCHV-based human vaccine directed against genital herpes orother sexually transmitted diseases that is to be administered to thevaginal mucosal epithelium with the intention of inducing residentimmunity, reference is made to Borgdorff et al. (2016) (Mucosal Immunol(2016) 9: 621-33). The latter publication reports that the KRT1, KRT4,KRT5, KRT6A, KRT10 and KRT13 genes are abundantly expressed in theepithelial layer of the vagina. Preferred promoters for use in RCCHVssuitable for the latter applications are those of the KRT1, KRT4 andKRT13 genes that are most selectively active and appear to haveessentially no activity in nerve ganglia.

TABLE 1 Expression of KRT genes Rel. transcript Rel. transcript levelover all Rel. transcript level in dorsal tissues- Gene ID level in skinroot ganglia median Mouse 16678 13452 4.7 4.6 KRT1 (epidermis) Human3848 6713 2.9 3.9 KRT1 Mouse 406220 37271 4.6 4.6 KRT77 Mouse 1666192700 158 124 KRT10 (epidermis) Human 3858 5939 15 93 KRT10 Data fromthe BioGPS database

For an RCCHV-based oncolytic therapy of human melanoma, in particularprimary melanoma, a suitable promoter may be that of the human MLANAgene or, possibly, that of the human TYR gene. Weinstein et al. (2014)(J Clin Aesthet Dermatol (2014) 7: 13-24). Both promoters typically haveelevated activity in skin melanomas (Human Protein Atlas;proteinatlas.org) but only minimal activity in nerve ganglia.

Tissue- or cell type-specific promoters for controlling the expressionof one or more replication-essential genes in an RCCHV of the presentdisclosure, including the promoters specifically disclosed above, areselected because they are active in the intended target cells butessentially inactive in cells of nerve ganglia, and their use in theherpesvirus recombinants is expected to preclude or minimize thepossibility of reactivation of the viruses from latency. It appearsimprobable that, when using such selective promoters, an unacceptablelevel of replication is detected in neural cells (which is a level thatenables detectable reactivation from latency in a subject). However, ifthis occurs, it is likely that the promoter concerned is alsoexcessively active in the target cells, i.e., that highly efficient(wildtype-like) replication could be had at a considerably lower levelof promoter activity. Hence, to reduce such undue viral replication inneural cells, it may be indicated to generally reduce the level ofexpression of the replication-essential gene(s) controlled by thepromoter. Various engineering approaches to achieve this goal are knownin the art. For example, protein-destabilizing elements could beintroduced into the replication-essential protein, e.g., near thecarboxy terminus of the protein. Well known sequence elements of thistype are the so-called PEST sequences that are thought to function asproteolytic signals. Rechsteiner and Rogers (1996) (Trends Biochem Sci(1996) 21: 267-71). These sequences contain regions enriched in proline(P), glutamate (E), serine (S) and threonine (T). PEST sequences arehydrophilic stretches of at least 12 amino acids in length, with theentire region flanked by lysine (K), arginine (R) or histidine (H), butnot interrupted by positively charged residues. RNA-destabilizingelements, AU-rich elements (ARE), may be added to the 3′UTR sequence ofthe replication-essential gene. Such elements were described in Zubiagaet al. (1995) (Mol Cell Biol (1995) 15: 2219-30). See also Matoulkova etal. (2012) RNA Biol 9: 563-76. RNA- and protein-destabilizing elementshave also been used in combination to dramatically reduce proteinlevels. Voon et al. (2005) Nucleic Acids Res 33 (3): e27. Otherapproaches are aimed at reducing translation efficiency. Theintroduction of highly stable secondary structure (hairpins) near the 5′end of the gene can dramatically reduce translation efficiency as shownby Babendure et al. (2006) (RNA (2006) 12: 851-61). Hence, suchsecondary structure elements could be introduced into thereplication-essential gene to reduce its expression.

The following description illustrates how an RCCHV of the presentdisclosure may be employed. The narrative focuses on vaccine uses. Howthe RCCHVs could be used for other therapeutic or prophylactic purposesshould also become readily apparent from this description.

In an exemplary vaccine application, a composition comprising aneffective amount of a heat-controlled or a heat- and SMR-controlledRCCHV of the present disclosure and, in the case of a heat- andSMR-controlled RCCHV, an effective amount of an SMR is administered to asubject intradermally or subcutaneously. Shortly after administration, aheating patch is activated and applied to the inoculation site region byeither the subject or the physician. Heating at about 43.5-45.5° C.(temperature of the patch surface in contact with the skin) will be fora period of about 10-60 min. The latter heat treatment will trigger onecycle of virus replication. If another round of replication is desired,another activated patch is applied to the inoculation site region at anappropriate later time. If an immunization procedure employing a heat-and SMR-controlled RCCHV involves sequential heat treatments, SMR mayalso need to be administered sequentially. Alternatively, a slow releaseformulation may be utilized that assures the presence of an effectiveconcentration of the SMR in the inoculation site region over the periodduring which viral replication is desired.

More generally, a body region to which an RCCHV of the presentdisclosure is administered, i.e., the inoculation site region, may beheated by any suitable method. Heat may be delivered or produced in thetarget region by different means including direct contact with a heatedsurface or a heated liquid, ultrasound, infrared radiation, or microwaveor radiofrequency radiation. As proposed in the above specific example,a practical and inexpensive solution may be offered by heating patches(or similar devices of other shapes, e.g., cylinders or cones, forheating mucosal surfaces of the nose, etc.) containing a supercooledliquid that can be triggered by mechanical disturbance to crystallize,releasing heat at the melting temperature of the chemical used. A usefulchemical may be sodium thiosulfate pentahydrate that has a meltingtemperature of about 48° C. U.S. Pat. Nos. 3,951,127, 4,379,448, and4,460,546. The technology is readily available and is already being usedin a number of health care products. That such heating patches arecapable of activating heat shock promoters in all human skin layers hasbeen verified experimentally. Voellmy et al. (2018) Cell StressChaperones 23(4): 455-466.

An “activating heat dose” is a heat dose that causes a transientactivation of heat shock transcription factor 1 (HSF1) in cells withinthe inoculation site region. Activation of HSF1 is evidenced by adetectably increased level of RNA transcripts of a heat-inducible heatshock gene over the level present in cells not exposed to the heat dose.Alternatively, it may be evidenced as a detectably increased amount ofthe protein product of such a heat shock gene. Moreover, an activatingheat dose may be evidenced by the occurrence of replication of aheat-controlled RCCHV, in the presence of an effective concentration ofan appropriate small-molecule regulator in the case of a heat- andSMR-controlled RCCHV.

An activating heat dose can be delivered to the target region at atemperature between about 41° C. and about 47° C. for a period ofbetween about 1 min and about 180 min. It is noted that heat dose is afunction of both temperature and time of exposure. Hence, similar heatdoses can be achieved by a combination of an exposure temperature at thelower end of the temperature range and an exposure time at the upper endof the time range, or an exposure temperature at the higher end of thetemperature range and an exposure time at the lower end of the timerange. Preferably, heat exposure will be at a temperature between about42° C. and about 46° C. for a duration of between about 5 min and about150 min. Most preferably, heat treatment is administered at atemperature between about 43.5° C. and about 45.5° C. for a duration ofbetween about 10 min and about 60 min. It is noted that it appearsfeasible to deliver an activating heat dose within a much shorter time,i.e., within seconds or even in the sub-second range, by intenseirradiation of the target region. Tolson and Roberts (2005) (Methods(2005) 35:149-157); Sajjadi et al. (2013) (Med Eng Phys (2013)35:1406-1414).

Concerning heat- and SMR-controlled RCCHVs of the present disclosure, anSMR should satisfy a number of criteria. Most important will be that thesubstance is safe; adverse effects should occur at most at an extremelylow rate and should be generally of a mild nature. Ideally, an SMR wouldbelong to a chemical group that is not used in human therapy. However,before any substance not otherwise developed for human therapy could beused as an SMR in a medical application of an RCCHV, it would have toundergo extensive preclinical and clinical testing. It may be moreefficacious to select a known and well-characterized drug substance thatis not otherwise administered to the specific population targeted fortreatment or immunization using an RCCHV. Alternatively, a known drugsubstance that will not need to be administered to subjects within atleast the first several weeks after RCCHV-mediated treatment orimmunization may be selected as an SMR. Thus, the potential low-levelrisk of disseminated replication of the RCCHV would be further reducedby the avoidance of administration of the drug substance during theperiod during which the RCCHV is systemically present. Subsequent,ideally sporadic, use of the drug substance under medical supervisionwill ensure that any significant inadvertent replication of an RCCHVwould be rapidly diagnosed and antiviral measures could be taken withoutdelay. In examples described herein, the SMR is a progesterone receptor(PR) antagonist or antiprogestin, e.g., mifepristone or ulipristal.Mifepristone and ulipristal fulfill the latter requirement of nottypically needing to be administered shortly after virus administration.Mifepristone and ulipristal have excellent human safety records.

An effective concentration of an SMR in the inoculation site region is aconcentration that enables replication (at least one round) of an RCCHVin infected cells of that region. What an effective concentration isdepends on the affinity of the SMR for its target transactivator. Howsuch effective concentration is achieved and for how long it ismaintained also depends on the pharmacokinetics of the particular SMR,which in turn depends on the route or site of administration of the SMR,the metabolism and route of elimination of the SMR, the subject to whichthe SMR is administered, i.e., the type of subject (human or othermammal), its age, condition, weight, etc. It further depends on the typeof composition administered, i.e., whether the composition permits animmediate release or a slow release of the regulator. For a number ofwell-characterized SMR-transactivator systems, effective concentrationsin certain experimental subjects have been estimated and are availablefrom the literature. This applies to systems based on progesteronereceptor, ecdysone receptors, estrogen receptors, and tetracyclinerepressor as well as to dimerizer systems, i.e., transactivatorsactivated by rapamycin or analogs (including non-immunosuppressiveanalogs), or FK506 or analogs. For example, an effective concentrationof mifepristone in rats can be reached by i.p. (intraperitoneal)administration of 5 μg mifepristone per kg body weight (5 μg/kg).Amounts would have to be approximately doubled (to about 10 μg/kg), ifthe SMR is administered orally. Wang, Y. et al. (1994) Proc Natl AcadSci USA 91: 8180-84. Amounts of an SMR that, upon administration by thechosen route to the chosen site, result in an effective concentrationare referred to as effective amounts of the SMR in question. How aneffective amount of an SMR that results in an effective concentrationcan be determined is well within the skills of an artisan.

In the afore-described specific example of how the novel immunizationmethod may be practiced, a heat- and SMR-controlled RCCHV of the presentdisclosure and an appropriate SMR were co-administered in a singlecomposition. RCCHV and SMR can also be administered in separatecompositions. Topical co-administration of immunizing virus and SMRappears advantageous for several reasons, including minimization ofpotential secondary effects of the SMR, further reduction of the alreadyremote possibility that virus may replicate systemically during theimmunization period, and minimization of the environmental impact ofelimination of SMR. Notwithstanding these advantages, the SMR may begiven by a systemic route, e.g., orally, which may be preferred if aformulation of the drug substance of choice is already available thathas been tested for a particular route of administration. The relativetiming of inoculation with RCCHV, administration of an appropriate heatdose and administration of an effective amount of SMR is derivative ofthe operational requirements of dual-responsive gene switch control.Typically, inoculation with immunizing virus will precede heattreatment. This is because heat activation of HSF1 is transient, andactivated factor returns to an inactive state within at most a few hoursafter activation. The dual-responsive transactivator gene present in theviral genome must be available for HSF1-mediated transcription duringthe latter short interval of factor activity. For the latter gene(s) tobecome available for transcription, the immunizing virus will have hadto adsorb to a host cell, enter the cell and unravel to present itsgenome to the cellular transcription machinery. Although not preferred,it is possible to heat-expose the inoculation site region immediatelyafter (or even shortly before) administration of the immunizing virus.Typically, the inoculation site region is heat-exposed at a time betweenabout 30 min to about 10 h after virus administration, although heattreatment may be administered even later. Regarding administration ofthe SMR, there typically will be more flexibility because it will bepossible to maintain an effective concentration systemically orspecifically in the inoculation site region for one to several days.Consequently, SMR can be administered prior to, at the time of orsubsequent to virus administration, the only requirement being that theregulator be present in an effective concentration in the inoculationsite region for the time needed for the target transactivator to fulfillits role in enabling viral replication. Typically, this time willcorrespond to that required for the completion of a round of inducedvirus replication. Typically, a round of virus replication will becompleted within about one day.

As has been alluded to before, in the novel immunization method an RCCHVmay be induced to replicate once or several times. Replication may bere-induced one to several days after the previous round of replication.Such repeated replication will serve to increase viral load in thesubject. For any round of replication to occur, the target cells thatare infected with RCCHV need to receive an activating heat dose and thetissue of which the latter cells are part (the inoculation site region)must contain an effective concentration of small-molecule regulator.

Inoculation can be by any suitable route. The body region (inoculationsite region) to which an RCCHV is administered may typically be acutaneous or subcutaneous region located anywhere on the trunk or theextremities of a subject. Preferably, administration of a composition ofthe invention comprising an RCCHV may be to a cutaneous or subcutaneousregion located on an upper extremity of the subject. Administration mayalso be to the lungs or airways, a mucous membrane in an orifice of asubject or any other tissue region in which the virus is capable ofreplicating.

The heat- and SMR-controlled RCCHVs exemplified herein are controlled bya heat- and antiprogestin-coactivated gene switch. They expressmifepristone- or ulipristal-activated chimeric transactivator GLP65 (orglp65). This transactivator comprises a DNA-binding domain from yeasttranscription factor GAL4, a truncated ligand-binding domain from ahuman progesterone receptor and a transactivation domain from the humanRelA protein (p65). Burcin et al. (1999); Ye et al. (2002). Otherexemplary SMR-activated transactivators than can be incorporated in anRCCHV include tetracycline/doxycycline-regulated tet-on repressors(Gossen & Bujard (1992) Proc Natl Acad Sci USA 89: 5547-51; Gossen etal. (1996) Science 268: 1766-69), and transactivators containing aligand-binding domain of an insect ecdysone receptor (No et al. (1996)Proc Natl Acad Sci USA 93: 3346-51). A stringently ligand-dependenttransactivator of this type is the RheoSwitch transactivator developedby Palli and colleagues (Palli et al. (2003) Eur J Biochem 270: 1308-15;Kumar et al. (2004) J Biol Chem 279: 27211-18). The RheoSwitchtransactivator can be activated by ecdysteroids such as ponasterone A ormuristerone A, or by synthetic diacylhydrazines such as RSL-1 (alsoknown as RH-5849). Dhadialla et al. (1998) Annu Rev Entomol 43: 545-69.Other SMR-activated transactivators may be used, provided that they canbe employed to control the activity of a target gene without alsocausing widespread deregulation of genes in cells of the intended hosts(subjects) and provided further that the associated SMRs have acceptablylow toxicity in the hosts at their effective concentrations.

A concern has been whether pre-existing immunity to a virus willpreclude its use as a vaccine or oncolytic vector. This issue not onlyrelates to viruses such as adenoviruses and herpesviruses that areendemic but also to viruses that not normally infect humans but are usedrepeatedly as vectors. There may have been more serious concernsregarding the effects of pre-existing immunity to adenovirus (type 5)than to any other vector. Draper & Heeney (2010) Nat Rev Microbiol 8:62-73. Steffensen et al. (2012) (PLoS ONE (2012) 7: e34884) demonstratedthat pre-existing immunity does not interfere with the generation ofmemory CD8 T cells upon vaccination with a heterologousantigen-expressing modified Ad5 vector, providing a basis for anefficient recall response and protection against subsequent challenge.Furthermore, the transgene product-specific response could be boosted byre-vaccination. The issue of pre-existing immunity to herpesviruses hasalso been examined in multiple studies. Brockman & Knipe (2002) J Virol76: 3678-87; Chahlavi et al. (1999) Gene Ther 6: 1751-58; Delman et al.(2000) Hum Gene Ther 11: 2465-72; Hocknell et al. (2002) J Virol 76:5565-80; Lambright et al. (2000) Mol Ther 2: 387-93; Herrlinger et al.(1998) Gene Ther 5: 809-19; Lauterbach et al. (2005 J Gen Virol 86:2401-10; Watanabe et al. (2007) Virology 357: 186-98. A majority ofthese studies reported little effect or only relatively minor effects onimmune responses to herpesvirus-delivered heterologous antigens or onanti-tumor efficacy of oncolytic herpesviruses. Brockman & Knipe (2002);Chahlavi et al. (1999); Delman et al. (2000); Hocknell et al. (2002);Lambright et al. (2000); Watanabe, D (2007). Two studies reportedsubstantial reductions of immune responses. Herrlinger (1998);Lauterbach et al. (2005). However, it appears that the results of thesestudies may not be generalized because compromised models were employed.One of the studies employed a tumor model that was only barelyinfectable with the mutant HSV strain used. Herrlinger (1998). The otherstudy employed a chimeric mouse immune model in combination with aseverely crippled HSV strain (ICP4⁻, ICP22⁻, ICP27−, vhs−) as the testvaccine. Lauterbach et al. (2005). All studies agreed that vaccine usesof herpesviruses are possible even in the presence of pre-existingimmunity. It may be added that pre-existing immunity, e.g., toherpesviruses, may not be of general concern for childhood preventativeor therapeutic interventions.

Herpesviruses have evolved a multitude of mechanisms for evading immunedetection and avoiding destruction. Tortorella et al. (2000) Annu RevImmunol 18: 861-926. Elimination or weakening of some of thesemechanisms could further enhance the potency of an RCCHV. For example,HSV-1 and HSV-2 express protein ICP47. This protein binds to thecytoplasmic surfaces of both TAP1 and TAP2, the components of thetransporter associated with antigen processing TAP. Advani & Roizman(2005) In: Modulation of Host Gene Expression and Innate Immunity byViruses (ed. P. Palese), pp. 141-61, Springer Verlag. ICP47 specificallyinterferes with MHC class I loading by binding to the antigen-bindingsite of TAP, competitively inhibiting antigenic peptide binding.Virus-infected human cells are expected to be impaired in thepresentation of antigenic peptides in the MHC class I context and,consequently, to be resistant to killing by CD8+ CTL. Deletion ordisablement of the gene that encodes ICP47 ought to significantlyincrease the potency of an RCCHV (both as an oncolytic agent and as avaccine).

The potency of an RCCHV may also be enhanced by including in the viralgenome an expressible gene for a cytokine or other component of theimmune system. A vaccination study in mice in whichreplication-defective herpesvirus recombinants expressing variouscytokines were compared demonstrated that virus-expressed IL-4 and IL-2had adjuvant effects. Osiorio & Ghiasi (2003) J Virol 77: 5774-83.Further afield, modulation of dendritic cell function by GM-CSF wasshown to enhance protective immunity induced by BCG and to overcomenon-responsiveness to a hepatitis B vaccine. Nambiar et al. (2009) Eur JImmunol 40: 153-61; Chou et al. (2010) J Immunol 185: 5468-75.

Expanding upon the basic definition given on p.8 as it relates tovaccine uses, an effective amount of an RCCHV of the present disclosureis an amount that upon administration to a subject and inducedreplication therein results in a detectably enhanced functional immunityof the subject (that is typically superior to the immunity induced by areplication-defective comparison virus or the unactivated RCCHV). Thisenhanced functional immunity may manifest itself as enhanced resistanceto infection or re-infection with a circulating (wild type) virus or mayrelate to enhanced suppression/elimination of a current infection.Hence, it may manifest itself by a reduced disease severity, diseaseduration or mortality subsequent to infection with said wild type virus.Alternatively, or in addition, in the case of an immunizing RCCHVexpressing a foreign (heterologous) antigen, immunity can relate topreventive or therapeutic immunity against pathogens expressing and/ordisplaying the latter foreign antigen. It is noted that a number offactors will influence what constitutes an effective amount of an RCCHV,including to some extent the site and route of administration of thevirus to a subject as well as the precise activation regimen utilized.Effective amounts of an RCCHV will be determined in dose-findingexperiments. Generally, for vaccine uses, an effective amount of anRCCHV of the present disclosure will be from about 102 to about 109plaque-forming units (pfu) of virus. More preferably, an effectiveamount will be from about 103 to about 108 pfu of virus, and even morepreferably from about 103 to about 107 pfu of virus. Larger amounts maybe indicated, in particular for oncolytic therapies.

A composition of the invention will comprise an effective amount of anRCCHV and, if an SMR is also administered as part of the composition, aneffective amount of the SMR. It further comprises, typically, apharmaceutically acceptable carrier or excipient. Although it may beadministered in the form of a fine powder, e.g., a lyophilizate, undercertain circumstances (see, e.g., U.S. Pat. Appl. Publ. No 20080035143;Chen et al. (2017) J Control Release 255: 36-44), a composition of theinvention typically is an aqueous composition comprising an RCCHV and,as the case may be, an SMR. It may be administered parenterally to asubject as an aqueous solution or, in the case of administration to amucosal membrane (e.g., airways), possibly as an aerosol thereof. See,e.g., U.S. Pat. No. 5,952,220. The term parenteral as used hereinincludes subcutaneous, intracutaneous (epidermis and/or dermis),intravenous, intramuscular, intraarticular, intraarterial,intrasynovial, intrasternal, intrathecal, intralesional and intracranialinjection or infusion techniques.

The compositions of the present invention will typically include abuffer component. The compositions will have a pH that is compatiblewith the intended use and is typically between about 6 and about 8. Avariety of conventional buffers may be employed such as phosphate,citrate, histidine, Tris, Bis-Tris, bicarbonate and the like andmixtures thereof. The concentration of the buffer generally ranges fromabout 0.01 to about 0.25% w/v (weight/volume).

The compositions of the invention comprising am RCCHV can furtherinclude, for example, preservatives, virus stabilizers, tonicity agentsand/or viscosity-increasing substances. As mentioned before, they mayalso include an appropriate SMR, or a formulation comprising such SMR.

Preservatives used in parenteral products include phenol, benzylalcohol, methyl paraben/propylparaben and phenoxyethanol. Phenoxyethanolappears to be the most widely used preservative found in vaccines.Preservatives are generally used in concentrations ranging from about0.002 to about 1% w/v. Meyer (2007) J Pharm Sci 96: 3155-67.Preservatives may be present in compositions comprising an RCCHV atconcentrations at which they do not or only minimally interfere with thereplication efficiency of the virus.

Osmolarity can be adjusted with tonicity agents to a value that iscompatible with the intended use of the compositions. For example, theosmolarity may be adjusted to approximately the osmotic pressure ofnormal physiological fluids, which is approximately equivalent to about0.9% w/v of sodium chloride in water. Examples of suitabletonicity-adjusting agents include, without limitation, chloride salts ofsodium, potassium, calcium and magnesium, dextrose, glycerol, propyleneglycol, mannitol, sorbitol and the like, and mixtures thereof.Preferably, the tonicity agent(s) will be employed in an amount toprovide a final osmotic value of 150 to 450 mOsm/kg, more preferablybetween about 220 to about 350 mOsm/kg and most preferably between about270 to about 310 mOsm/kg.

If indicated, the compositions of the present disclosure can furtherinclude one or more viscosity-modifying agents such as cellulosepolymers, including hydroxypropylmethyl cellulose, hydroxyethylcellulose, ethylhydroxyethyl cellulose, hydroxypropyl cellulose, methylcellulose, carboxymethyl cellulose, glycerol, carbomers, polyvinylalcohol, polyvinyl pyrrolidone, alginates, carrageenans, guar, karaya,agarose, locust bean, tragacanth and xanthan gums. Such viscositymodifying components are typically employed in an amount effective toprovide the desired degree of thickening. Viscosity-modifying agents maybe present in compositions comprising an RCCHV at concentrations atwhich they do not or only minimally interfere with the replicationefficiency of the virus.

If the composition also contains an SMR, an effective amount of such SMRcan be included in the composition in the form of a powder, solution,emulsion or particle. As also provided before, an effective amount of anSMR to be co-delivered with an effective amount of an RCCHV will be anamount that yields an effective concentration of the SMR in theinoculation site region, which effective concentration enables at leastone round of replication of the RCCHV in infected cells of that region.To maintain an SMR at an effective concentration for a more extendedperiod, it may be included in the form of a slow-release formulation(see also below).

Methods for amplifying herpesviruses are well known in the laboratoryart. Industrial scale-up has also been achieved. Hunter (1999) J Virol73: 6319-26; Rampling et al. (2000) Gene Ther 7: 859-866; Mundle et al.(2013) PLoS ONE 8(2): e57224. Various methods for purifying viruses havebeen disclosed. See, e.g., Mundle et al. (2013) and references citedtherein; Wolf and Reichl (2011) Expert Rev Vaccines 10: 1451-75.

While an SMR can be co-administered with an RCCHV in a singlecomposition, a composition comprising an RCCHV and a compositioncomprising an SMR can also be administered separately. The lattercomposition will comprise an effective amount of an SMR formulatedtogether with one or more pharmaceutically acceptable carriers orexcipients.

A composition comprising an SMR may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir, preferably by oraladministration, administration by injection or deposition at the site ofvirus inoculation. The compositions may contain any conventionalnon-toxic, pharmaceutically acceptable carrier, adjuvant or vehicle. Insome cases, the pH of the formulation may be adjusted withpharmaceutically acceptable acids, bases or buffers to enhance thestability of the formulated SMR or its delivery form.

Liquid dosage forms of an SMR for oral administration includepharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition, the liquid dosage formsmay contain inert diluents commonly used in the art such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can also include, e.g., wetting agents, emulsifying andsuspending agents, sweetening, flavoring, and perfuming agents.

Injectable preparations, for example sterile injectable aqueous oroleaginous suspensions, may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic, parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose, any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of an SMR, it may be desirable to slowthe absorption of the compound from, e.g., subcutaneous, intracutaneousor intramuscular injection. This may be accomplished by the use of aliquid suspension of crystalline or amorphous material with poor watersolubility. The rate of absorption of the SMR then depends upon its rateof dissolution, which, in turn, may depend upon crystal size andcrystalline form. Alternatively, delayed absorption of a parenterallyadministered SMR is accomplished by dissolving or suspending thecompound in an oil vehicle. Injectable depot forms are made by formingmicrocapsule matrices of the compound in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of compound topolymer and the nature of the particular polymer employed, the rate ofcompound release can be controlled. Examples of other biodegradablepolymers include poly(orthoesters) and poly(anhydrides). Depotinjectable formulations are also prepared by entrapping the compound inliposomes or microemulsions that are compatible with body tissues.

Compositions for rectal or vaginal administration can be suppositorieswhich can be prepared by mixing the SMR with suitable non-irritatingexcipients or carriers such as cocoa butter, polyethylene glycol or asuppository wax which are solid at ambient temperature but liquid atbody temperature and therefore melt in the rectum or vaginal cavity andrelease the SMR.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the SMR ismixed with at least one inert, pharmaceutically acceptable excipient orcarrier such as sodium citrate or dicalcium phosphate and/or: a) fillersor extenders such as starches, lactose, sucrose, glucose, mannitol, andsilicic acid, b) binders such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c)humectants such as glycerol, d) disintegrating agents such as agar-agar,calcium carbonate, potato or tapioca starch, alginic acid, certainsilicates, and sodium carbonate, e) solution-retarding agents such asparaffin, f) absorption accelerators such as quaternary ammoniumcompounds, g) wetting agents such as, for example, cetyl alcohol andglycerol monostearate, h) absorbents such as kaolin and bentonite clay,and i) lubricants such as talc, calcium stearate, magnesium stearate,solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof.In the case of capsules, tablets and pills, the dosage form may alsocomprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the SMR only, or preferentially, in acertain part of the intestinal tract, optionally, in a delayed manner.Examples of embedding compositions that can be used include polymericsubstances and waxes.

Dosage forms for topical, intradermal or transdermal administration ofan SMR include ointments, pastes, creams, lotions, gels, powders,solutions, sprays, inhalants or patches. The SMR is admixed understerile conditions with a pharmaceutically acceptable carrier and anypreservatives or buffers as may be required.

The ointments, pastes, creams and gels may contain, in addition to anSMR, excipients such as animal and vegetable fats, oils, waxes,paraffins, starch, tragacanth, cellulose derivatives, polyethyleneglycols, silicones, bentonites, silicic acid, talc and zinc oxide, ormixtures thereof.

Powders and sprays can contain, in addition to the SMR, excipients suchas lactose, talc, silicic acid, aluminum hydroxide, calcium silicatesand polyamide powder, or mixtures of these substances. Sprays canadditionally contain customary propellants.

Transdermal patches have the added advantage of providing controlleddelivery of a compound. Such dosage forms can be made by dissolving ordispensing the compound in the proper medium. Absorption enhancers canalso be used to increase the flux of the compound into or across theskin.

For pulmonary delivery, a composition comprising an effective amount ofan SMR is formulated and administered to the subject in solid or liquidparticulate form by direct administration e.g., inhalation into therespiratory system. Solid or liquid particulate forms of the SMRprepared for practicing the present invention include particles ofrespirable size: that is, particles of a size sufficiently small to passthrough the mouth and larynx upon inhalation and into the bronchi andalveoli of the lungs. Delivery of aerosolized therapeutics, particularlyaerosolized antibiotics, is known in the art (see, for example U.S. Pat.Nos. 5,767,068 and 5,508,269, and WO 98/43650). A discussion ofpulmonary delivery of antibiotics is also found in U.S. Pat. No.6,014,969.

What an effective amount of an SMR is will depend on the activity of theparticular SMR employed, the route of administration, time ofadministration, the stability and rate of excretion of the particularSMR as well as the nature of the specific composition administered. Itmay also depend on the age, body weight, general health, sex and diet ofthe subject, other drugs used in combination or contemporaneously withthe particular SMR employed and like factors well known in the medicalarts.

Ultimately, what is an effective amount of an SMR can be determined indose-finding experiments, in which replication of a heat- andSMR-controlled RCCHV is assessed experimentally in the inoculation siteregion. Once an effective amount has been determined in animalexperiments, it may be possible to estimate a human effective amount.“Guidance for Industry. Estimating the maximum safe starting dose forinitial clinical trials for therapeutics in adult healthy volunteers”,U.S. FDA, Center for Drug Evaluation and Research, July 2005,Pharmacology and Toxicology. For example, as estimated from rat data, aneffective human amount of orally administered mifepristone (for enablingat least one cycle of virus replication) will be between about 1 andabout 100 μg/kg body weight.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. The singular encompasses the plural, unlessotherwise stated or clearly contradicted by context.

The description herein of any aspect or embodiment of the inventionusing terms such as reference to an element or elements is intended toprovide support for a similar aspect or embodiment of the invention that“consists of”,” “consists essentially of” or “substantially comprises”that particular element or elements, unless otherwise stated or clearlycontradicted by context (e. g., a composition described herein ascomprising a particular element should be understood as also describinga composition consisting of that element, unless otherwise stated orclearly contradicted by context).

This invention includes all modifications and equivalents of the subjectmatter recited in the aspects or claims presented herein to the maximumextent permitted by applicable law.

The present invention, thus generally described, will be understood morereadily by reference to the following examples, which are provided byway of illustration and are not intended to be limiting of the presentinvention.

EXAMPLES Example 1: Isolation and Characterization of Mouse KRT1 andKRT77 Promoters

KRT1 and KRT77 promoter and RNA leader sequences were PCR-amplified frommouse genomic DNA using a standard protocol. Promoter sequences lieupstream from the start of transcription site (−), and RNA leadersequences begin at the start of transcription site (+). PCRamplification employed either primers mKRT1F1(5″-CTGACTGGCTTTAGCCCCTT-3′) (SEQ ID NO: 1) and mKRT1R1(5′-GCCTTAGAGAGAGGTGAGAGC-3′) (SEQ ID NO: 2), or primers mKRT1F2(5′-GCCACAAAACACTTTCAGGTACATA-3′) (SEQ ID NO: 3) and mKRT1R2(5″-TGATGCCTTAGAGAGAGGTGA-3′) (SEQ ID NO: 4). For KRT77, the primerswere mKRT77F1 (5″-AAGATTTATTAGTGCGTTTTGGTGC-3′) (SEQ ID NO: 5) andmKRT77R1 (5″-CAGAAGCACTGGTAGCAAGGA-3′) (SEQ ID NO: 6). The latter primersequences were designed based on published mKRT1 and mKRT77 sequences(FIGS. 1 and 2 ) (SEQ ID NOS: 7 and 8). The amplified KRT1 and KRT77segments were then subcloned into vector pGL4.16 (Promega Corp.,Madison, Wis.) that contains a promoter-less luciferase reporter geneluc2CP from Photinus pyralis. To achieve this, the amplified KRT1 andKRT77 DNAs were further amplified with forward and reverse primers thatalso contained a Kpnl or a BamHI recognition sequence, respectively. There-amplified DNAs were digested with Kpnl and BamHI, and the fragmentswere gel-purified and then ligated into Kpnl/BgII-double-digestedpGL4.16. Following transformation, colonies were picked and expanded,and inserts were subjected to nucleotide sequence analysis. Clone KRT11.2 contains mKRT1 sequences from position −993 to position +56, cloneKRT1 2.3 mKRT1 sequences from position −1026 to position +60 and cloneKRT77 3.2 mKRT77 sequences from position −986 to position +72.

The ability of the KRT promoters to drive transcription from thefunctionally linked luciferase reporter gene was assessed in severalcell types. Since expression was to be compared between different celltypes which may differ in transfectability and general transcriptionalactivity, the different KRT constructs were co-transfected with aconstruct that contained a β-galactosidase reporter gene controlled bythe ubiquitously active ROSA promoter (pDRIVE-mROSA; InvivoGen Corp.).Transfection of subconfluent cultures grown under standard conditionsemployed a standard lipofectamine procedure. The activities of the KRTpromoters were expressed as ratios of relative luciferase (LUC) toβ-galactosidase (B-GAL) activities. LUC activity was measured using theDual Glo Luciferase Assay System (Promega) and BGAL activity using theBeta-Glo Assay System (Promega).

TABLE 2 Activity of KRT promoters in Neuro2a cells Rel. LUC activity-pGL4.16 Rel. B-GAL LUC/ Rel. LUC vector activity (− (B-GAL × Constructactivity background background) 10⁻⁴) KRT1 1.2 70.5 +/− 7.3  13.7130073.7 +/− 1.0 6589 KRT1 2.3 91.0 +/− 12.0 34.2 136262.3 +/− 2.5 3389KRT77 3.2 55.3 +/− 29.6 0 131378.7 +/− 0 6127 pGL4.16 56.8 +/− 36.8112490.0 +/− (promoter-less) 5862

TABLE 3 Activity of KRT promoters in HEK293T cells Rel. LUC activity-pGL4.16 Rel. B-GAL LUC/ Rel. LUC vector activity (− (B-GAL × Constructactivity background background) 10⁻⁴) KRT1 1.2 9500.9 +/− 8400.8 36006.0+/− 2333.5 1029 3995.9 KRT1 2.3 6051.6 +/− 4951.5 41178.5 +/− 1201.81486 1267.1 KRT77 3.2 20990 +/− 19889.9 42632.5 +/− 4668.9 2313 3459.5pGL4.16 1100.1 +/− 36409.2 +/− (promoter-less) 172 8205.9

Results from two representative experiments are shown in Tables 2 and 3.The experiment in Table 2 was conducted employing mouse neural cell lineNeuro2a, and the experiment in Table 3 with the human epithelial cellline HEK293T. The results demonstrate clearly that the KRT1 and KRT77promoters are highly active in the epithelial cells but essentiallyinactive in the neural cells. Therefore, the isolated promoter segmentscontain all information required for their cell type-specific activity.

Example 2: Construction of Heat- and Antiprogestin-Controlled RCCHVsHSV-GS1 and HSV-GS3

The generation of the viral recombinants was performed by homologousrecombination of engineered plasmids along with purified virion DNA intorabbit skin cells (RS) (or HEK293T cells in other Examples) by thecalcium phosphate precipitation method as previously described. Bloom(1998) HSV (Methods Mol Med (1998) 10: 369-386). All plasmids used toengineer the insertions of heat shock promoters (HSP70B promoters in theexample constructions described herein), transactivators or theGAL4-responsive promoters for recombination into the HSV-1 genome werecloned from HSV-1 strain 17syn+. Plasmid IN994 was created as follows:an HSV-1 upstream recombination arm was generated by amplification ofHSV-1 DNA (from base pairs 95,441 to 96,090) with DB112 (5′GAG CTC ATCACC GCA GGC GAG TCT CTT3′) (SEQ ID NO: 9) and DB113 (5′GAG CTC GGT CTTCGG GAC TAA TGC CTT3′) (SEQ ID NO: 10). The product was digested withSacl and inserted into the Sacl restriction site of pBluescript tocreate pUP. An HSV-1 downstream recombination arm was generated usingprimers DB115-Kpnl (5′GGG GTA CCG GTT TTG TTT TGT GTG AC3′) (SEQ ID NO:11) and DB120-Kpnl (5′GGG GTA CCG GTG TGT GAT TTC GC3′) (SEQ ID NO: 12)to amplify HSV-1 genomic DNA sequence between base pairs 96,092 and96,538. The PCR product was digested with Kpnl, and cloned into Kpnldigested pUP to create pIN994, which recombines with HSV-1 at theintergenic UL43/44 region.

HSV-GS1

HSV-GS1 contains a transactivator (TA) gene cassette inserted into theintergenic region between UL43 and UL44. In addition, the ICP4 promoterhas been replaced with a GAL4-responsive promoter (GAL4-bindingsite-containing minimal promoter) in both copies of the short repeats. Afirst recombination plasmid pIN:TA1 was constructed by inserting a DNAsegment containing a GLP65 gene under the control of a promoter cassettethat combined a human HSP70B (heat shock) promoter and a GAL4-responsivepromoter (described in Vilaboa et al. (2005)) into the multiple cloningsite of plasmid pIN994, between flanking sequences of the HSV-1 UL43 andUL44 genes. The TA cassette was isolated from plasmid Hsp70/GAL4-GLP65(Vilaboa et al. (2005)) and was cloned by 3-piece ligation to minimizethe region that was amplified by PCR. For the left insert,Hsp70/GAL4-GLP65 was digested with BamHI and BstX1 and the resulting2875 bp band was gel-purified. This fragment contains the Hsp70/GAL4promoter cassette as well as the GAL4 DNA-binding domain, theprogesterone receptor ligand-binding domain and part of the p65activation domain of transactivator GLP65. The right insert wasgenerated by amplifying a portion of pHsp70/GAL4-GLP65 with the primersTA.2803-2823.fwd (5′TCG ACA ACT CCG AGT TTC AGC3′) (SEQ ID NO: 13) andBGHpA.rev (5′ CTC GCG GCC GCA TCG ATC CAT AGA GCC CAC CGC ATC C3′) (SEQID NO: 14). The 763 bp PCR product was digested with BstX1 and NotI, andthe resultant 676 bp band was gel-purified. This band contained the3′end of the p65 activation domain and the BGHpA. For the vector, pIN994was digested with BamHI and NotI, and the resulting 4099 bp fragment wasgel-purified and shrimp alkaline phosphatase (SAP)-treated. The twoinserts were then simultaneously ligated into the vector, creating anintact TA cassette. Subsequent to transformation, colony #14 wasexpanded, and the plasmid was verified by restriction enzyme analysisand then by sequence analysis.

One μg of pIN:TA1 was co-transfected with 2 μg of purified HSV-1 (17+)virion DNA into RS cells by calcium phosphate precipitation. Theresulting pool of viruses was screened for recombinants by pickingplaques, amplifying these plaques on 96 well plates of RS cells, anddot-blot hybridization with a ³²P-labeled DNA probe prepared by labelinga TA fragment by random-hexamer priming. A positive well was re-plaquedand re-probed 5 times and verified to contain the TA by PCR and sequenceanalysis. This intermediate recombinant was designated HSV-17GS43.

A second recombination plasmid, pBS-KS:GAL4-ICP4, was constructed thatcontained a GAL4-responsive promoter inserted in place of the nativeICP4 promoter by cloning it in between the HSV-1 ICP4 recombination armsin the plasmid pBS-KS:ICP4Δpromoter. This placed the ICP4 transcriptunder the control of the heterologous GAL4 promoter. This particularpromoter includes six copies of the yeast GAL4 UAS (upstream activatingsequence), the adenovirus E1b TATA sequence and the synthetic intronIvs8. This promoter was excised from the plasmid pGene/v5-HisA(Invitrogen Corp.) with AatII and HindIII, and the resulting 473 bpfragment was gel-purified. For the vector, pBS-KS:ICP4Δ promoter wasdigested with AatII and HindIII and the resulting 3962 bp fragmentgel-purified and SAP-treated. Ligation of these two fragments placed theGAL4 promoter in front of the ICP4 transcriptional start-site.Subsequent to transformation, colony #5 was expanded, test-digested andverified by sequencing.

One μg of pBS-KS:GAL4-ICP4 was co-transfected with 4 μg of purifiedHSV-17GS43 virion DNA into cells of the ICP4-complementing cell line E5(DeLuca and Schaffer (1987) Nucleic Acids Res 15: 4491-4511) by calciumphosphate precipitation. The resulting pool of viruses was screened forrecombinants by picking plaques, amplifying these plaques on 96 wellplates of E5 cells, and dot-blot hybridization with a ³²P-labeled DNAprobe prepared by labeling the GAL4-responsive promoter fragment byrandom-hexamer priming. A positive well was re-plaqued and re-probed 7times and verified to contain the GAL4-responsive promoter in bothcopies of the short repeat sequences by PCR and sequence analysis. Thisrecombinant was designated HSV-GS1.

To obtain pBS-KS:ΔSacl, the Sacl site was deleted from the polylinker ofplasmid vector pBluescript-KS+, by digesting the plasmid with Sacl. Theresulting 2954 bp fragment was gel-purified, treated with T4 DNApolymerase to produce blunt ends, re-circularized and self-ligated.Recombination plasmid BS-KS:ICP4Δ promoter was constructed as follows:to generate a first insert, cosmid COS48 (a gift of L. Feldman) wassubjected to PCR with the primers HSV1.131428-131404 (5′ CTC AAG CTT CTCGAG CAC ACG GAG CGC GGC TGC CGA CAC G3′) (SEQ ID NO: 15) andHSV1.130859-130880 (5′ CTC GGT ACC CCA TGG AGG CCA GCA GAG CCA GC3′)(SEQ ID NO: 16). The primers placed HindIII and XhoI sites on the 5′ endof the region and NcoI and Kpnl sites on the 3′ end, respectively. The600 bp primary PCR product was digested with HindIII and Kpnl, and theresulting 587 bp fragment was gel-purified. Vector pBS-KS:ΔSacl wasdigested with HindIII and Kpnl, and the resulting 2914 bp fragment wasgel-purified and SAP-treated. Ligation placed the first insert into thevector's polylinker, creating pBS-KS:ICP4-3′end. To generate a secondinsert, cosmid COS48 was subjected to PCR with the primersHSV1.132271-132250 (5′ CTC GCG GCC GCA CTA GTT CCG CGT GTC CCT TTC CGATGC3′) (SEQ ID NO: 17) and HSV1.131779-131800 (5′ CTC GAG AAG CTT ATGCAT GAG CTC GAC GTC TCG GCG GTA ATG AGA TAC GAG C3′) (SEQ ID NO: 18).These primers placed NotI and Spel sites on the 5′ end of the region andAatII, Sacl, Nsil, HindIII and XhoI sites on the 3′ end, respectively.The 549 bp primary PCR product was digested with NotI and XhoI, and theresulting 530 bp band was gel-purified. This fragment also contained the45 bp OriS hairpin. Plasmid BS-KS:ICP4-3′ end was digested with NotI andXhoI and the resulting 3446 bp band was gel-purified and SAP-treated.Ligation generated pBS-KS:ICP4Δ promoter. The inserts in pBS-KS:ICP4Δpromoter were verified by sequence analysis.

HSV-GS3 contains a transactivator (TA) gene cassette inserted into theintergenic region between UL43 and UL44. In addition, the ICP4 promoterhas been replaced with a GAL4-responsive promoter (GAL4-bindingsite-containing minimal promoter) in both copies of the short repeats.Furthermore, the ICP8 promoter was replaced with a GAL4-responsivepromoter. The construction of this recombinant virus involved placing asecond HSV-1 replication-essential gene (ICP8) under control of aGAL4-responsive promoter. HSV-GS1 was used as the “backbone” for theconstruction of this recombinant. ICP8 recombination plasmidpBS-KS:GAL4-ICP8 was constructed. This plasmid contained aGAL4-responsive promoter inserted in place of the native ICP8 promoterby cloning it in between the HSV-1 ICP8 recombination arms in theplasmid pBS-KS:ICP8Δpromoter. This placed the ICP8 transcript under thecontrol of the heterologous GAL4-responsive promoter. ThisGAL4-responsive promoter was excised from the plasmid pGene/v5-HisA(Invitrogen Corp.) with AatII and HindIII, and the resulting 473 bpfragment was gel-purified. For the vector, pBS-KS:ICP8Δpromoter wasdigested with AatII and HindIII, and the resulting 4588 bp fragmentgel-purified and SAP-treated. Ligation of the latter two DNA fragmentsplaced the GAL4-responsive promoter cassette in front of the ICP8transcriptional start-site. Subsequent to transformation, colony #10 wasexpanded, test-digested and verified by sequencing.

One μg of pBS-KS:GAL4-ICP8 was co-transfected with 10 μg of purifiedHSV-GS1 virion DNA into E5 cells by calcium phosphate precipitation.Subsequent to the addition of mifepristone to the medium, thetransfected cells were exposed to 43.5° C. for 30 minutes and thenincubated at 37° C. Subsequently, on days 2 and 3, the cells were againincubated at 43.5° C. for 30 minutes and then returned to 37° C. Plaqueswere picked and amplified on 96 well plates of E5 cells in mediasupplemented with mifepristone. The plates were incubated at 43.5° C.for 30 minutes 1 hour after infection and then incubated at 37° C.Subsequently, on days 2 and 3, the plates were also shifted to 43.5° C.for 30 minutes and then returned to 37° C. After the wells showed90-100% CPE, the plates were dot-blotted and the dot-blot membranehybridized with a ³²P-labeled DNA probe prepared by labeling the HSV-1ICP8 promoter fragment that was deleted. A faintly positive well wasre-plaqued and re-probed 8 times and verified to have lost the ICP8promoter and to contain the GAL4-responsive promoter in its place by PCRand sequence analysis. This recombinant was designated HSV-G53.

Recombination plasmid pBS-KS:ICP8Δpromoter was constructed usingessentially the same strategy as that described above for the creationof pBS-KS:ICP4Δ promoter: a first insert was PCR-amplified from HSV-117syn+ virion DNA using the primers HSV1.61841-61865 (5′ CTC AGA ACC CAGGAC CAG GGC CAC GTT GG3′) (SEQ ID NO: 19) and HSV1.62053-62027 (5′ CTCATG GAG ACA AAG CCC AAG ACG GCA ACC3′) (SEQ ID NO: 20) and subcloned toyield intermediate vector pBS-KS:ICP8-3′ end. A second insert wassimilarly obtained using primers HSV1.62173-62203 (5′ CTC GGA GAC CGGGGT TGG GGA ATG AAT CCC TCC3′) (SEQ ID NO: 21) and HSV1.62395-62366 (5′CTC GCG GGG CGT GGG AGG GGC TGG GGC GGA CC3′) (SEQ ID NO: 22) and wassubcloned into pBS-KS:ICP8-3′ end to yield pBS-KS:ICP8Δpromoter.

Example 3: Construction of Heat-Controlled RCCHVs: HSV-GS51-52

HSV-GS51 and HSV-GS52 contain ICP4 genes that are controlled by a humanHSP70B promoter. Furthermore, the promoter of the UL38/VP19c gene isreplaced with the mouse KRT77 promoter in HSV-GS51 and the mouse KRT1promoter in HSV-GS52, respectively.

HSV-GS51

HSV-1 wildtype strain syn+ is used as the “backbone” for theconstruction of this recombinant. ICP4 recombination plasmidpBS-KS:HSP70B-ICP4 is constructed that contains a human HSP70B promoterinserted in place of the native ICP4 promoter by cloning it in betweenthe HSV-1 ICP4 recombination arms in the plasmid pBS-KS:ICP4Δ promoter.To isolate a human HSP70B promoter fragment, construct p17 is digestedwith BamHI, ends are filled in by Klenow DNA polymerase, and the DNA isfurther digested with HindIII. A 0.45 kbp (kbp=1,000 bp) promoterfragment is gel-purified (Voellmy et al. (1985) Proc. Natl. Acad. Sci.USA 82: 4949-53). For the vector, pBS-KS:ICP4Δ promoter is digested withZral and HindIII. The resulting 3.96 kbp fragment is gel-purified.Ligation of the latter two DNA fragments places the HSP70B promoter infront of the ICP4 transcriptional start-site. Subsequent totransformation, several colonies are expanded, and plasmid DNAssubjected to restriction and then sequence analysis to identifypBS-KS:HSP70B-ICP4.

To obtain pBS-KS:ΔSacl, the Sacl site was deleted from the polylinker ofplasmid vector pBluescript-KS+, by digesting the plasmid with Sacl. Theresulting 2.95 kbp fragment was gel-purified, treated with T4 DNApolymerase to produce blunt ends, re-circularized and self-ligated.Recombination plasmid BS-KS:ICP4Δ promoter was constructed as follows:to generate a first insert, cosmid COS48 (a gift of L. Feldman) wassubjected to PCR with the primers HSV1.131428-131404 (5′ CTC AAG CTT CTCGAG CAC ACG GAG CGC GGC TGC CGA CAC G3′) (SEQ ID NO: 15) andHSV1.130859-130880 (5′ CTC GGT ACC CCA TGG AGG CCA GCA GAG CCA GC3′)(SEQ ID NO: 16). The primers placed HindIII and XhoI sites on the 5′ endof the region and NcoI and Kpnl sites on the 3′ end, respectively. The0.60 kbp primary PCR product was digested with HindIII and Kpnl, and theresulting 0.59 kbp fragment was gel-purified. Vector pBS-KS:ΔSacl wasdigested with HindIII and Kpnl, and the resulting 2.91 kbp fragment wasgel-purified and SAP-treated. Ligation placed the first insert into thevector's polylinker, creating pBS-KS:ICP4-3′end. To generate a secondinsert, cosmid COS48 was subjected to PCR with the primersHSV1.132271-132250 (5′ CTC GCG GCC GCA CTA GTT CCG CGT GTC CCT TTC CGATGC3′) (SEQ ID NO: 17) and HSV1.131779-131800 (5′ CTC GAG AAG CTT ATGCAT GAG CTC GAC GTC TCG GCG GTA ATG AGA TAC GAG C3′) (SEQ ID NO: 18).These primers placed NotI and Spel sites on the 5′ end of the region andAatII, Sacl, Nsil, HindIII and XhoI sites on the 3′ end, respectively.The 0.55 kbp primary PCR product was digested with NotI and XhoI, andthe resulting 0.53 kbp band was gel-purified. This fragment alsocontained the 45 bp OriS hairpin. Plasmid BS-KS:ICP4-3′ end was digestedwith NotI and XhoI and the resulting 3.45 kbp band was gel-purified andSAP-treated. Ligation generated pBS-KS:ICP4Δ promoter. The inserts inpBS-KS:ICP4Δ promoter were verified by sequence analysis.

One μg of pBS-KS:HSP70B-ICP4 is co-transfected with 10 μg of purifiedHSV-1 syn+ virion DNA into cells of the ICP4-complementing cell line E5(DeLuca, N. A. and Schaffer, P. A. (1987)) by calcium phosphateprecipitation. The resulting pool of viruses is screened forrecombinants by picking plaques, amplifying these plaques on 96 wellplates of E5 cells, and dot-blot hybridization with a ³²P-labeled DNAprobe prepared by labeling the HSP70B promoter fragment byrandom-hexamer priming. A positive well is re-plaqued and re-probedseveral times and verified to contain the HSP70B promoter in both copiesof the short repeat sequences by PCR and sequence analysis. Thisintermediary recombinant is designated HSV-17GS51.

To place the UL38/VP19c gene under regulation of a mouse KRT77 promoter,plasmid pBS-KS:KRT77-UL38 is constructed as follows. Plasmid KRT77 3.2is subjected to PCR amplification using primers mKRT77AF1(5″-GGACTGACGTCAAGATTTATTAGTGCGTTT TGGTGC-3′) (SEQ ID NO: 23) andmKRT77AR1 (5″-CAACCCGGGCAGAAGCACTGGT AGCAAGGA-3′) (SEQ ID NO: 24). Theamplified fragment containing KRT77 sequences from position −986 toposition +72 is digested with AatII and Smal and is gel-purified. Forthe vector, plasmid pBS-KS:UL38Δpromoter containing HSV-1 UL38recombination arms is digested with HindII, ends are filled in usingKlenow DNA polymerase, and the DNA is further digested with AatII. Theresulting 4.28-kbp fragment is gel-purified and SAP-treated. The lattertwo fragments are ligated. Following transformation, several coloniesare amplified and tested for the presence of KRT77 sequences by dot blotusing a ³²P-labeled KRT77 probe. A clone containing the complete KRT77promoter and RNA leader sequence of pKRT77 3.2 as assessed by nucleotidesequence analysis is designated pBS-KS:KRT77-UL38. PlasmidpBS-KS:UL38Δpromoter was constructed by deletion of the region from −1to −47 of the UL38 promoter, i.e., by synthesizing two PCR fragments(one 0.44 kbp and the other 0.55 kbp long) on either side of thedeletion and cloning these into pBS II KS+.

To produce recombinant HSV-GS51, HEK293T cells are co-transfected with 1μg of plasmid pBS-KS:KRT77-UL38 and 10 μg of purified HSV-17GS51 virionDNA by calcium phosphate precipitation. The transfected cells areexposed to 43.5° C. for 30 min and then incubated at 37° C.Subsequently, on days 2 and 3, the cells are again incubated at 43.5° C.for 30 min and then returned to 37° C. Plaques are picked and amplifiedon 96-well plates of RS. One hour after infection, the plates areincubated at 43.5° C. for 30 min and then further incubated at 37° C.Subsequently, on days 2 and 3, the plates are also shifted to 43.5° C.for 30 min and then returned to 37° C. After the wells show 90 to 100%cytopathic effect, the plates are dot blotted, and the dot blot membraneis hybridized with a ³²P-labeled DNA probe prepared by labeling themouse KRT77 promoter segment. A positive well is re-plaqued andre-probed several times, and is verified to have lost the UL38 promoterand to contain the KRT77 promoter in its place by PCR and sequenceanalysis. This recombinant is designated HSV-GS51. It is noted that forrecombination and isolation of HSV-GS51 E5 cells previously transfectedwith a VP19c-expression plasmid such as pUL38FBpCl (Adamson et al.(2006) J Virol 80: 1537-1548) may be employed instead of HEK293T cells,circumventing the need for repeated heat treatments for virusamplification.

HSV-GS52

To place the UL38/VP19c gene under regulation of a mouse KRT1 promoter,plasmid pBS-KS:KRT1-UL38 is constructed as follows. Plasmid KRT1 1.2 issubjected to PCR amplification using primers mKRT1AF1(5″-GGACTGACGTCTGACTGGCTTTAGCCCCTT-3″) (SEQ ID NO: 25) and mKRT1AR1(5″-CAACCCGG GCCTTAGAGAGAGGTGAGAGC-3″) (SEQ ID NO: 26). The amplifiedfragment containing KRT1 sequences from position −993 to position +56 isdigested with AatII and Smal and is gel-purified. For the vector,plasmid pBS-KS:UL38Δpromoter is digested with HindII, ends are filled inusing Klenow DNA polymerase, and the DNA is further digested with AatII.The resulting 4.28-kbp fragment is gel-purified and SAP-treated. Thelatter two fragments are ligated. Following transformation, severalcolonies are amplified and tested for the presence of KRT1 sequences bydot blot using a ³²P-labeled KRT1 probe. A clone containing the completeKRT1 promoter and leader sequence of pKRT1 1.2 as assessed by nucleotidesequence analysis is designated pBS-KS:KRT1-UL38.

HEK293T cells are co-transfected with 1 μg of plasmid pBS-KS:KRT1-UL38and 10 μg of purified HSV-17GS51 virion DNA by calcium phosphateprecipitation. The transfected cells are incubated and heat-treated, andplaques are picked and amplified as described for the construction ofHSV-GS51. Dot blots are hybridized with a ³²P-labeled KRT1 promoter DNAprobe. A positive well is re-plaqued and re-probed several times, and isverified to have lost the UL38 promoter and to contain the KRT1 promoterin its place by PCR and sequence analysis. This recombinant isdesignated HSV-GS52.

Example 4: Construction of Heat-Controlled RCCHVs Expressing aTransactivator: HSV-GS53-54

HSV-GS53 contains ICP4 and ICP8 genes that are controlled by aGAL4-hHSF1 transactivator driven by a human HSP70B promoter. In HSV-GS54the expression of the same viral genes is controlled by a GAL4-hHSF1transactivator that is driven by a human HSP70B/GAL4 promoter cassette(i.e., a sequence acting as both a heat shock promoter and atransactivator-responsive promoter). Furthermore, the promoter of theUL38/VP19c gene is replaced with the mouse KRT1 promoter in bothrecombinants.

HSV-GS53

Plasmid CMV-hHSF1 contains in between the HindIII and EcoR1 sites ofvector pcDNA3.1 a human HSF1 cDNA fragment that includes the entire 529residues-long HSF1-coding sequence as well as upstream and downstreamuntranslated sequences (FIG. 3 ) (SEQ ID NO: 27). Baler et al. (1993)Mol Cell Biol 13: 2486-2496; Xia et al. (1999) Cell Stress Chaperon 4:8-18. The single NotI site in pCMV-hHSF1 is destroyed by NotI digestion,filling-in using the Klenow fragment of DNA polymerase I,self-religation, transformation and isolation of a colony that lacks theNotI site. This plasmid is designated CMV-hHSF1-delNotI. A segmentcontaining the sequence coding for hHSF1 residues 431-529 (encompassingthe hHSF1 activation domain), 3′nontranslated sequences of hHSF1 and theBGHpA region (present in the cDNA3.1 vector) is PCR-amplified frompCMV-hHSF1-delNotI using primers HSF1F (5′ GACGGTACCCCGACCTTGACAGCAGCCTG) (SEQ ID NO: 28) and BGHpA.rev (5′ CTCCTCGCGGCCGCATCGATCCATAGAGCCCACCGCATCC) (SEQ ID NO: 14). The amplified fragment isdigested with Kpnl and NotI. Vector pSG424 containing an expressiblegene for a GAL4 DNA-binding domain (residues 1-147) is digested withHindIII and Kpnl to release the GAL4(1-147)-encoding fragment that isgel-purified. This fragment and the above Kpnl/NotI-digested PCRfragment from pCMV-hHSF1-delNotI are co-ligated intoHindIII/Not-double-digested and SAP-treated vector pBlueScript II SK.The resulting plasmid that contains the GAL4-HSF1 transactivator-codingsequence is designated pGAL4/HSF1TA. Plasmid GAL4/HSF1TA is digestedwith HindIII and NotI, and the released transactivator-encoding fragment(1.48 kbp in length) is gel-purified. Construct p17 is digested withBamHI and HindIII. A 045 kbp human HSP70B promoter fragment isgel-purified (Voellmy et al. (1985)). The latter two fragments areco-ligated with a pIN994 BamHI/NotI vector fragment. (pIN994 is digestedwith BamHI and NotI, and the resulting 4.10 kbp fragment is gel-purifiedand SAP-treated.) The resulting recombination plasmid is designated pIN:HSP-TA. Plasmid IN994 was created as described under Example 2.

One μg of pIN: HSP-TA is co-transfected with 10 μg of purified HSV-GS3virion DNA into E5 cells by calcium phosphate precipitation. Thetransfected cells are exposed to 43.5° C. for 30 minutes and thenincubated at 37° C. Subsequently, on days 2 and 3, the cells are againincubated at 43.5° C. for 30 minutes and then returned to 37° C. Plaquesare picked and amplified on 96 well plates of E5 cells. The plates areincubated at 43.5° C. for 30 minutes 1 hour after infection and thenincubated at 37° C. Subsequently, on days 2 and 3, the plates are alsoshifted to 43.5° C. for 30 minutes and then returned to 37° C. After thewells show 90-100% CPE, the plates are dot-blotted and the dot-blotmembrane hybridized with a ³²P-labeled DNA probe prepared by labeling a0.71 kbp Ball/EcoRI fragment from pCMV-hHSF1 (containing sequencescoding for HSF1 residues 437-529 and 3′untranslated sequences). Astrongly positive well is re-plaqued and re-probed several times andverified to have lost the HSP70/GAL4-GLP65 transactivator cassette andto contain in its place the HSP70B promoter-GAL4-HSF1 transactivatorcassette by PCR and sequence analysis. This recombinant is designatedHSV-17GS53.

One μg of pBS-KS:KRT1-UL38 is co-transfected with 10 μg of purifiedHSV-17GS53 virion DNA into HEK293T cells by calcium phosphateprecipitation. The transfected cells are incubated and heat treated, andplaques are picked and amplified as described for the construction ofHSV-GS51. Dot blots are hybridized with a ³²P-labeled KRT1 promoter DNAprobe. A positive well is re-plaqued and re-probed several times, and isverified to have lost the UL38 promoter and to contain the KRT1 promoterin its place by PCR and sequence analysis. This recombinant isdesignated HSV-G553.

HSV-GS54

This recombinant is constructed by the procedure described for HSV-G553with the sole exception that the 0.45 kbp BamHI/HindIII HSP70B promoterfragment is substituted by a 1.06-kbp BamHI/HindIII HSP70B/GAL4 promoterfragment isolated from pHsp70/GAL-fLuc (Vilaboa et al. (2005)).

Example 5: Construction of Heat- and Antiprogestin-Controlled RCCHVsHSV-GS1A and HSV-GS3A

Recombinant HSV-GS1 contains, inserted in the intergenic region betweenUL43 and UL44, a GLP65 transactivator (TA) gene that is under the (dual)control of an HSP70 and a GAL4-responsive promoter. In addition, theICP4 promoter is replaced with a GAL4-responsive promoter (GAL4-bindingsite-containing minimal promoter) in both copies of the short repeats.In HSV-GS3, derived from HSV-GS1, the ICP8 promoter is also replacedwith a GAL4-responsive promoter. In HSV-GS1A and HSV-GS3A, derived fromHSV-GS1 and HSV-GS3 respectively, the UL38/VP19c gene is controlled by amouse KRT1 promoter.

To produce recombinants HSV-GS1A or HSV-GS3A, HEK293T cells areco-transfected by calcium phosphate precipitation with 1 μg ofpBS-KS:KRT1-UL38 (described under Example 3) and 10 μg of purifiedHSV-GS1 virion DNA (for HSV-GS1A) or HSV-GS3 virion DNA (for HSV-GS3A).Subsequent to the addition of mifepristone (10 nM) to the medium, thetransfected cells are exposed to 43.5° C. for 30 minutes and thenincubated at 37° C. Subsequently, on days 2 and 3, the cells are againincubated at 43.5° C. for 30 minutes and then returned to 37° C. Plaquesare picked and amplified on 96 well plates of HEK293T cells in mediasupplemented with mifepristone. The plates are incubated at 43.5° C. for30 minutes 1 hour after infection and then incubated at 37° C.Subsequently, on days 2 and 3, the plates are also shifted to 43.5° C.for 30 minutes and then returned to 37° C. After the wells show 90-100%CPE, the plates are dot-blotted and the dot-blot membrane hybridizedwith a ³²P-labeled KRT1 promoter DNA probe. A positive well isre-plaqued and re-probed several times and verified to have lost theUL38 promoter and to contain the KRT1 promoter in its place by PCR andsequence analysis. This recombinant is designated HSV-GS1A or 3A,respectively.

Example 6: Construction of Heat- and Antiprogestin-Controlled RCCHVHSV-GS3B

Recombinant HSV-GS3B contains inserted in the intergenic region betweenUL43 and UL44, a GLP65 transactivator (TA) gene that is under the (dual)control of an HSP70 and a GAL4-responsive promoter. In addition, theICP4 and ICP8 promoters are replaced with a GAL4-responsive promoter(GAL4-binding site-containing minimal promoter). The UL38/VP19c gene iscontrolled by a mouse KRT1 promoter. Furthermore, the US12 gene ismutated to render its protein product (ICP47) nonfunctional.

ICP47 amino acid residue K31 was changed to G31, and R32 to G32. Neumannet al. (1997) J. Mol. Biol. 272: 484-492; Galocha et al. (1997) J. Exp.Med. 185: 1565-1572. A 500-bp ICP47-coding sequence-containing fragmentwas PCR-amplified from virion DNA of strain 17syn+. The fragment wasPCR-amplified as two pieces (a “left-hand” and a “right-hand” piece),using two primer pairs. The mutations were introduced through the 5′ PCRprimer for the right-hand fragment. The resulting amplified left-handand mutated right-hand fragments were subcloned into vector pBS, and thesequence in subclones was confirmed by sequence analysis. A subclonecontaining the 500-bp fragment with the desired mutations in ICP47codons 31 and 32 was termed pBS:mut-ICP47.

One μg of pBS:mut-ICP47 was co-transfected with 10 μg of purifiedHSV-GS3 virion DNA into E5 cells by calcium phosphate precipitation.Subsequent to the addition of mifepristone to the medium, thetransfected cells were exposed to 43.5° C. for 30 minutes and thenincubated at 37° C. Subsequently, on days 2 and 3, the cells were againincubated at 43.5° C. for 30 minutes and then returned to 37° C. Plaqueswere picked and amplified on 96 well plates of E5 cells in mediasupplemented with mifepristone. The plates were incubated at 43.5° C.for 30 minutes 1 hour after infection and then incubated at 37° C.Subsequently, on days 2 and 3, the plates were also shifted to 43.5° C.for 30 minutes and then returned to 37° C. After the wells showed90-100% CPE, the plates were dot-blotted and the dot-blot membranehybridized with a ³²P-labeled oligonucleotide probe to the mutated ICP47region. A positive well was re-plaqued and re-probed several times andverified by sequence analysis to contain the expected mutated ICP47 genesequence. This recombinant was designated HSV-GS4.

To produce recombinant HSV-GS3B, HEK293T cells are co-transfected bycalcium phosphate precipitation with 1 μg of pBS-KS:KRT1-UL38 (describedunder Example 3) and 10 μg of purified HSV-GS4 virion DNA. Subsequent tothe addition of mifepristone (10 nM) to the medium, the transfectedcells are exposed to 43.5° C. for 30 minutes and then incubated at 37°C. Subsequently, on days 2 and 3, the cells are again incubated at 43.5°C. for 30 minutes and then returned to 37° C. Plaques are picked andamplified on 96 well plates of HEK293T cells in media supplemented withmifepristone. The plates are incubated at 43.5° C. for 30 minutes 1 hourafter infection and then incubated at 37° C. Subsequently, on days 2 and3, the plates are also shifted to 43.5° C. for 30 minutes and thenreturned to 37° C. After the wells showed 90-100% CPE, the plates aredot-blotted and the dot-blot membrane hybridized with a ³²P-labeled KRT1promoter DNA probe. A positive well is re-plaqued and re-probed severaltimes and verified to have lost the UL38 promoter and to contain theKRT1 promoter in its place by PCR and sequence analysis. Thisrecombinant is designated HSV-GS3B.

Example 7: Construction of Heat- and Antiprogestin-Controlled RCCHVHSV-GS3C

Recombinant HSV-GS3C contains inserted in the intergenic region betweenUL43 and UL44, a GLP65 transactivator (TA) gene that is under thecontrol of an HSP70/GAL4 promoter cassette. In addition, the ICP4 andICP8 promoters are replaced with GAL4-responsive promoters (GAL4-bindingsite-containing minimal promoters), and the UL38/VP19c gene iscontrolled by a mouse KRT77 promoter. Furthermore, the recombinantcontains an insertion between the UL37 and UL38 genes of a gene cassetteexpressing the EIV Prague/56 hemagglutinin (HA) gene driven by the CMVIE promoter.

Recombinant HSV-GS11 was derived from the vector HSV-GS3 and contains aninsertion between the UL37 and UL38 genes of a gene cassette expressingthe A/Equine/Prague/1/56 (H7N7) hemagglutinin (HA) gene driven by theCMV IE promoter. The recombination plasmid was constructed by thefollowing sequential steps. First, the 814 bp fragment containing theregion spanning the HSV-1 UL37/UL38 intergenic region from nt83,603-84,417 from the plasmid NK470 was subcloned into pBS SK+ that hadhad the MCS removed (digestion with Kpnl/Sacl) to yield pBS:UL37/38. Acassette containing a synthetic CMV IE promoter flanked by the pBS-SK+MCS was ligated into pBS:UL37/38 that was digested with BspE1/AfIII,which cuts between the UL37 and UL38 genes to yield the plasmidpIN:UL37/38. The EIV Prague/56 HA gene was PCR-cloned from cDNA preparedfrom EIV Prague/56. Briefly, RNA was prepared by Trizol extraction of astock of EIV Prague/56 and reverse-transcribed using Omni-Script ReverseTranscriptase (Qiagen) according to the manufacturer's instructions. ThecDNA was subcloned into pBS, and a clone containing the HA gene(pBS-EIVPrague56/HA) was confirmed by sequence analysis. The Prague/56HA gene was excised from this plasmid and inserted behind the CMVpromoter in the plasmid pIN:UL37/38 to yield plasmidpIN:37/38-Prague56/HA. To produce recombinant HSV-GS11, RS cells wereco-transfected with plasmid pIN:37/38-Prague56/HA and purified HSV-GS3virion DNA. Subsequent to the addition of mifepristone to the medium,the co-transfected cells were exposed to 43.5° C. for 30 min and thenincubated at 37° C. Subsequently, on days 2 and 3, the cells were againincubated at 43.5° C. for 30 min and then returned to 37° C. Picking andamplification of plaques, screening and plaque purification wasperformed essentially as described for HSV-GS3. The resultingplaque-purified HSV-GS11 was verified by Southern blot as well as by PCRand DNA sequence analysis of the recombination junctions.

To produce recombinant HSV-GS3C, HEK293T cells are co-transfected bycalcium phosphate precipitation with 1 μg of pBS-KS:KRT77-UL38(described under Example 3) and 10 μg of purified HSV-GS11 virion DNA.Subsequent to the addition of mifepristone (10 nM) to the medium, thetransfected cells are exposed to 43.5° C. for 30 minutes and thenincubated at 37° C. Subsequently, on days 2 and 3, the cells are againincubated at 43.5° C. for 30 minutes and then returned to 37° C. Plaquesare picked and amplified on 96 well plates of HEK293T cells in mediasupplemented with mifepristone. The plates are incubated at 43.5° C. for30 minutes 1 hour after infection and then incubated at 37° C.Subsequently, on days 2 and 3, the plates are also shifted to 43.5° C.for 30 minutes and then returned to 37° C. After the wells show 90-100%CPE, the plates are dot-blotted and the dot-blot membrane hybridizedwith a ³²P-labeled KRT77 promoter DNA probe. A positive well isre-plaqued and re-probed several times and verified to have lost theUL38 promoter and to contain the KRT77 promoter in its place by PCR andsequence analysis. This recombinant is designated HSV-GS3C.

Example 8: Analysis of Differential and Regulated Replication of RCCHVsin Epithelial and Neural Cells

Stocks of RCCHVs HSV-GS51-54, HSV-GS1A, and HSV-GS3A-C can be propagatedin HEK393T cells. Infected cultures are subjected to daily heattreatment at 43.5 (43-44) ° C. for 30 min for three successive days. ForHSV-GS1A and HSV-GS3A-C, the medium is supplemented with 10 nMmifepristone.

Replication of an RCCHV and wildtype viral strain 17syn+ is compared inthe epithelial cell line HEK293T and the neural cell line Neuro2a.HEK293T cells are grown in complete Dulbecco's modified Eagle's medium,supplemented with 10% fetal bovine serum and 2 mM I-glutamine andNeuro2a cells in in Eagle's Minimum Essential Medium plus 10% FBS. Theability of HSV-1 to infect and replicate in HEK293T and Neuro-2a cellshas been documented previously. Zhao et al. (2016) Cell Host & Microbe20: 770-784; Parker et al. (2000) Proc Natl Acad Sci USA 97: 2208-2213.

Confluent monolayers of target cells in multiple parallel plates areinfected with virus (RCCHV or wildtype virus 17syn+) at a multiplicityof infection (MOI) of 3. Virus is allowed to adsorb for 1 h at 37° C.,and then the inoculum is removed, and the cells are overlayed withmedium. For cultures infected with an HSV-GS1A or HSV-GS3A-Crecombinant, mifepristone treatment (10 nM) is initiated at the time ofthe initial infection or is omitted. Heat treatment is performed eitherimmediately after infection or 4 h later by floating the sealed dishesin a 43.5° C. water bath for 30 min, or the heat treatment is omitted.The dishes are then incubated at 37° C. At 0, 4 or 8, 12 or 16, and 24or 28 h postinfection, two dishes are removed, and the cells are scrapedinto medium for harvesting and subjected to two freeze-thaw cycles.Infectious virus is then determined by titrating the lysate of each dishin triplicate on 24-well plates of confluent E5 cells transfected 24 hprior to infection with expression plasmids pICP8 and pUL38FBpCl(Adamson et al. (2006)) using Lipofectamine 2000 (Life Technologies).Plaques are visualized after 2 days by staining with crystal violet.Alternatively, plaques are visualized 2 days after infection using anantibody plaque assay, essentially as previously described (Bewig andSchmidt (2000) Biotechniques 28: 870-873). Briefly, medium is removed,and the monolayers fixed with cold 100% methanol for 20 min at 20° C.The monolayers are then washed with phosphate-buffered saline (PBS) and,subsequent to addition of 100 μl of a 1/1000 dilution of a polyclonalanti-HSV antiserum (rabbit anti-HSV HRP conjugate, DAKO labs), areincubated for 1 h a room temperature. The antiserum is aspirated, themonolayers are rinsed twice with PBS, and the plaques are visualized byadding 200 μl of Immunopure DAB substrate (Pierce Chemicals), which isremoved after approximately 10 min by rinsing with PBS.

To construct expression plasmid pICP8, a 3777 bp fragment containing theentire ICP8-coding sequence and the native promoter of the ICP8 gene wasPCR-amplified from HSV-1 17syn+ virion DNA using primersHSV1.58409-58444 (CTCCTCTTTATTTTACACACATTCCCC GCCCCGCCCTAGGTT) (SEQ IDNO: 29) and HSV1.62186-62150 CTCCTCAACCCCGG TCTCCAACCCTCCCCTTGACCGTCGCCG(SEQ ID NO: 30) and subcloned into pBS-KS:ΔSacl. The plasmid insert wasverified by sequence analysis. To obtain pBS-KS:ΔSacl, the Sacl site wasdeleted from the polylinker of plasmid vector pBluescript-KS+, bydigesting the plasmid with Sacl.

Representative results of growth experiments of the kind described above(but testing different recombinant viruses in different cell types) weredisclosed, e.g., in Bloom et al. (2015) (FIGS. 2 and 3 ). In theinstantly described growth experiments, recombinant RCCHVs of thepresent disclosure are expected replicate with similar efficiency aswildtype virus 17syn+ in the epidermal cells that had been heat-treatedand, in the case of HSV-GS1A or HSV-GS3A-C had been exposed tomifepristone. Essentially no replication of RCCHVs should be apparent incultures that were not heat-treated or, in the case of HSV-GS1A orHSV-GS3A-C viruses had not also been exposed to mifepristone. In theneural cells, in which 17syn+ will replicate well, replication of RCCHVsshould not be observed, regardless of whether the cultures wereheat-treated and/or exposed to mifepristone.

Example 9: Reactivation from Latency

Herpesviruses are known to latently infect sensory nerve cells in whichthey can reactivate under adverse circumstances, e.g., during a highfever. In the RCCHVs of the present disclosure the replication-essentialUL38 gene is controlled by a KRT1 or KRT77 promoter, which promoters areessentially inactive in nerve cells. Hence, reactivation of RCCHVs insensory neurons should not occur in a subject, even if the subject isexperiencing a high fever or other severe stress and, in the case of anHSV-GS1A or HSV-GS3A-C recombinant, is concurrently administered anantiprogestin. This assumption is tested in a well-established mousemodel of heat-induced reactivation (Sawtell and Thompson (1992) J Virol66: 2150-2156). In this model, all mice that had been inoculated on thelightly abraded rear footpads with HSV-1 wildtype virus strain 17syn+were found to contain latent virus in their ganglia at 30 days afterinoculation. No spontaneous reactivation was observed. Heat treatmentreactivated virus in the dorsal root ganglia (DRG) in about 80% oflatently infected animals.

In an exemplary experiment to demonstrate that RCCHV HSV-GS3A does notreactivate, 4 groups (n=10) of 4- to 6-week-old Swiss Webster mice areinoculated on the abraded rear footpads with 250 pfu of wildtype HSV-1strain 17syn+(2 groups) or 50,000 pfu of HSV-GS3A (2 groups). Thirtydays later, all animals will receive mifepristone (0.5 mg/kg)intraperitoneally, and one of the 17syn+ groups and one of the HSV-GS3Bgroups are subjected to heating in a 43° C. water bath as described inSawtell and Thompson (1992). Individual animals or small groups areslowly lowered into the water. Body temperature increase is monitored bymeans of a rectally inserted thermocouple. Heat treatments are for 10min (with body temperature reaching 43° C. after about 7 min). The miceare then allowed to recover at 37° C. for 15 min. Twenty-four hourslater, all animals are euthanized. Dorsal root ganglia (DRG) arerecovered, homogenized and subjected to a freeze-thaw cycle to lysecells. After clarification, the extracts are used to infect confluentcultures of pICP8- and pUL38FBpCl-co-transfected E5 cells. The culturesare then monitored daily for cytopathic effects (reactivation).

Example 10: Vaccination Against Herpes Disease—an Immunization/ChallengeExperiment

Previous immunization/challenge experiments demonstrated thatimmunization with replication-competent controlled viruses such asHSV-GS3 induced a strong protective response against challenge withwildtype herpesvirus. The following exemplary experiment is performed todemonstrate that RCCHVs of the present disclosure are similarly capableof protecting against disease caused by a subsequent herpesvirusinfection. Induction of protective immunity is evaluated in a mousefootpad lethal challenge model (McKendall (1977) Infect Immun 16:717-719). Viruses HSV-GS3A, HSV-GS3 and KD6 (an ICP4⁻replication-incompetent HSV-1 recombinant (Dobson et al. 1990. Neuron5:353-360) or vehicle are administered under anesthesia to the plantarsurfaces of both rear feet of adult Swiss Webster outbred female mice(50,000 PFU per animal; 20 animals per group). Concurrently, and again24 h later, the animals of one of the HSV-GS3A groups and one of theHSV-GS3 groups will receive an intraperitoneal injection of 50 μg/kg ofbody weight ulipristal (an antiprogestin). Three hours afterinoculation, the mice in these groups are subjected to heat treatment(44.5° C. for 10 min) by immersion of their hind feet in atemperature-controlled water bath. Three weeks later, all animals willbe reinoculated with 50,000 PFU/animal of the recombinant virus they hadreceived before (or with vehicle for the mock group). The animals in theHSV-GS3A and HSV-GS3 groups will be exposed to ulipristal and subjectedto heat treatment as before. Three weeks later, all the animals arechallenged with a 20-fold lethal dose of the HSV-1 wild-type strain17syn+ administered by the same route as the immunizing viruses.Survival of the animals is followed until no more lethal endpoints arereached, i.e., until all surviving animals have fully recovered. As isknown from our previous experiments, replication-defective virus KD6will induce a modest level of immunity. Unactivated HSV-GS3 will providea comparable degree of protection. In contrast, activated (i.e.,heat-treated in the presence of antiprogestin) HSV-GS3 will producecomplete or near complete protective effect. It is expected thatHSV-GS3A will perform essentially as HSV-GS3.

Example 11: Vaccination Against Influenza Disease—anImmunization/Challenge Experiment

Previous immunization/challenge experiments demonstrated thatimmunization with replication-competent controlled viruses such asHSV-GS11 induced a strong protective response against challenge withwildtype influenza virus. The following exemplary experiment isperformed to demonstrate that RCCHVs of the present disclosureexpressing an antigen from another pathogen are capable of protectingagainst disease caused by infection with that pathogen. As doesHSV-GS11, RCCV HSV-GS3C expresses a hemagglutinin (HA) gene frominfluenza virus A/Equine/Prague/1/56 (H7N7).

Viruses HSV-GS3C, HSV-GS11 and HSV-GS3 (negative control) or vehicle areadministered under anesthesia to the plantar surfaces of both rear feetof adult female BALB/c mice (250,000 PFU virus per animal; 20 animalsper group). Concurrently, and again 24 h later, the animals will receivean intraperitoneal injection of 50 μg/kg of body weight ulipristal.Three hours after inoculation, the mice are subjected to heat treatment(44.5° C. for 10 min) by immersion of their hind feet in atemperature-controlled water bath. Three weeks later, all animals willbe reinoculated with 250,000 PFU/animal of the recombinant virus theyhad received before (or vehicle) as well as will be exposed toulipristal and subjected to heat treatment as before. Three weeks later,all animals are challenged with a several-fold lethal dose ofA/Equine/Prague/1/56 that is administered intranasally. Animals areobserved until no more lethal endpoints (>20% weight loss) are reached,i.e., until all surviving animals are recovering. As is known from ourprevious experiments, HSV-GS11 will provide essentially completeprotection against the influenza virus challenge, and HSV-GS3 will haveat most a marginal protective effect. It is expected that HSV-GS3C willperform essentially as HSV-GS11.

Generally known molecular biology and biochemistry methods are/wereused. Molecular biology methods are described, e.g., in “Currentprotocols in molecular biology”, Ausubel, F. M. et al., eds., John Wileyand Sons, Inc. ISBN: 978-0-471-50338-5.

1. The replication-competent controlled alpha-herpesvirus of claim 16,wherein the replication-competent controlled alpha-herpesvirus is arecombinant alpha-herpesvirus comprising (a) a first heterologouspromoter that is a nucleic acid sequence that acts as a heat shockpromoter, the first heterologous promoter controlling the expression ofa first replication-essential gene of the replication-competentcontrolled alpha-herpesvirus, and (b) a second heterologous promoterthat is known to be active in cells in an inoculation site region of amammalian subject to which region the replication-competent controlledalpha-herpesvirus is to be administered but is known to be essentiallyinactive in cells of nerve ganglia of the mammalian subject, the secondheterologous promoter being functionally linked to a secondreplication-essential gene of the replication-competent controlledalpha-herpesvirus.
 2. The replication-competent controlledalpha-herpesvirus according to claim 16, wherein the inoculation site inregion is a cutaneous or subcutaneous region, or a mucosal membrane. 3.The replication-competent controlled alpha-herpesvirus of claim 1,wherein the first heterologous promoter is functionally linked to thefirst replication-essential gene of the replication-competent controlledalpha-herpesvirus.
 4. The replication-competent controlledalpha-herpesvirus of claim 1, wherein the first heterologous promoter isfunctionally linked to a gene for a heterologous transactivator that hasbeen inserted in the genome of the replication-competent controlledalpha-herpesvirus and the first replication-essential gene isfunctionally linked to a transactivator-responsive promoter.
 5. Thereplication-competent controlled alpha-herpesvirus of claim 4, whereinthe first heterologous promoter is a nucleic acid sequence that acts asa heat shock promoter as well as a transactivator-responsive promoter.6. The replication-competent controlled alpha-herpesvirus of claim 4,wherein the heterologous transactivator is a small-moleculeregulator-activated transactivator.
 7. The replication-competentcontrolled alpha-herpesvirus of claim 6, wherein the small-moleculeregulator-activated transactivator contains a truncated ligand-bindingdomain from a progesterone receptor and is activated by an antiprogestinor other molecule capable of interacting with the ligand-binding domainand of activating the transactivator.
 8. The replication-competentcontrolled alpha-herpesvirus of claim 16, wherein thereplication-competent controlled alpha-herpesvirus is a virus selectedfrom the group consisting of an HSV-1, an HSV-2 and a varicella zostervirus.
 9. The replication-competent controlled alpha-herpesvirus ofclaim 16, wherein the replication-competent controlled alpha-herpesvirusis an HSV-1 or HSV-2 and is lacking a functional ICP47 gene.
 10. Thereplication-competent controlled alpha-herpesvirus of claim 16 furthercomprising at least one of an expressed gene from another pathogen, anexpressed heterologous gene encoding an immune-modulatory polypeptideand an expressed heterologous gene encoding another polypeptide.
 11. Thereplication-competent controlled alpha-herpesvirus of claim 10, whereinthe expressed gene from another pathogen is a gene encoding an influenzavirus surface antigen or internal protein or parts thereof.
 12. Thereplication-competent controlled alpha-herpesvirus of claim 10, whereinthe expressed gene from another pathogen is a gene encoding a humanimmunodeficiency virus surface antigen or internal protein or partsthereof.
 13. A vaccine composition comprising an effective amount of thereplication-competent controlled alpha-herpesvirus of claim 16 and apharmaceutically acceptable carrier or excipient.
 14. Use of thereplication-competent controlled alpha-herpesvirus of claim 16 forpreventative or therapeutic vaccination against diseases caused by thevirus from which the replication-competent controlled alpha-herpesviruswas constructed.
 15. Use of the replication-competent controlledalpha-herpesvirus of claim 16 further comprising an expressed gene fromanother pathogen for preventative or therapeutic vaccination againstdiseases caused by that other pathogen.
 16. A replication-competentcontrolled alpha-herpesvirus whose replication can be transientlyactivated in infected nonneural cells but that cannot be so activated ininfected sensory or other neural cells.
 17. The replication-competentcontrolled alpha-herpesvirus of claim 1, wherein the second heterologouspromoter is selected from the promoters of a KRT1, a KRT4, a KRT5, aKRT6A, a KRT10, a KRT11, a KRT13, a KRT77, an MLANA and a TYR gene.